Rna interference using a universal target

ABSTRACT

The present invention provides novel methods for manipulating levels of expression of gene products using RNA interference (RNAi). The methods disclosed can be used to investigate gene function, to create disease-resistant organisms, and to treat disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/680,449, filed Oct. 6, 2003 (now U.S. Pat. No. 7,422,853), whichclaims benefit under 35 U.S.C. § 119(e) of U.S. Application Ser. No.60/416,353, filed Oct. 4, 2002; the disclosures of both of which arehereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention generally relates to the field of research intogene product function, and also disease etiology. It describes a methodfor altering the levels of expression of gene products that can be usedin cells, both in culture and in situ.

BACKGROUND OF THE INVENTION

The interference of expression of specific genes following theintroduction of double-stranded RNAs (dsRNAs) of corresponding sequencehas been observed in a variety of organisms. Initially described as thepost-transcriptional gene silencing (PTSG) of transgenes in transgenicplants, similar dsRNA-dependent gene silencing has now been observed inprotozoa, fungi, nematodes, insects, and mammals, and the phenomenon isnow generally referred to as “RNA interference.”

RNA interference (RNAi), which has been defined as the “the process ofsequence-specific, post-transcriptional gene silencing in animals andplants, initiated by double-stranded RNA (dsRNA) that is homologous insequence to the silenced gene” (Elbashir, et al., Nature 411: 494-498(2001)), has also been described as “the process whereby dsRNA inducesthe sequence-specific degradation of homologous mRNA” (Chiu & Rana.Molecular Cell 10:549-561 (2002)). Many of the mechanistic details ofRNAi first came to light from studies in which long dsRNAs matching thesequences of specific target gene transcripts were introduced into thenematode worm, Caenorhabditis elegans (Fire et al., Nature 391:806-811(1998)). These early studies inspired many more experiments to beconducted in a variety of organisms, and it is now clear that homologousmachinery for RNAi is widely distributed among eukaryotic organisms,including mammals. Although initial attempts to provoke RNAi in mammalsand mammalian cells with long dsRNAs failed, it was later determined thefailures occurred because such RNA molecules activate an antiviralresponse that leads to a general inhibition of translation andultimately cell death. Subsequent studies in a variety of non-mammalianspecies demonstrated that long dsRNAs introduced into cells areenzymatically cleaved into shorter duplexes comprising two complementarysingle-stranded RNAs of ˜21-25 nucleotides (see Sharp, Genes & Dev.15:485-490 (2001), and references therein). More recent studies havedemonstrated that, while dsRNAs of 30 basepairs or more elicit theaforementioned antiviral response, RNA duplexes comprising twocomplementary single-strands of 21 nucleotides each, which pair to forma 19 basepair duplexed region with two nucleotide 3′ overhangs (socalled small or short interfering RNAs (siRNAs)), can mediate RNAi incultured mammalian cells without evoking an antiviral response (Elbashiret al., Nature 411:494-498 (2001)). Additionally, when appropriatelytargeted via their nucleotide sequence, these siRNAs can specificallysuppress the expression of both endogenous genes and heterologoustransgenes, of corresponding sequence. Even more recent studies havedemonstrated that while double-stranded siRNAs are very effective atmediating RNAi in a variety of cell types, short, single-stranded,hairpin-shaped RNAs can also mediate RNAi, presumably because they areprocessed into siRNAs by cellular enzymes (Sui et al., Proc. Natl. Acad.Sci. U.S.A. 99:5515-5520 (2002); Yu et al., Proc. Natl. Acad. Sci.U.S.A. 99:6047-6052 (2002); and Paul et al., Nature Biotech. 20:505-508(2002)). This discovery has significant and far-reaching implicationssince the production of such small hairpin RNAs (shRNAs) can be readilyachieved in vivo by transfecting cells with transcription vectorsbearing short inverted repeats separated by a small number of (e.g.,six) nucleotides. Additionally, if features are included to ensure thestability of the transcription plasmid, or direct the integration of thetranscription cassette into the host cell genome, the RNAi induced bythe encoded shRNAs, can be made stable and heritable.

Since it was first exploited to silence specific genes in C. elegans,RNAi has become an irreplaceable tool for molecular, cellular anddevelopmental biologists seeking to discover the functions of specificgenes. Although few of the molecular mechanisms of RNAi are known indetail, it is clear that the degradative process involves the assemblyof a multisubunit ribonucleoprotein nuclease complex, known as RISC (forRNA-induced silencing complex), which is somehow guided by the antisensestrand of an siRNA to a complementary target sequence in a mature RNAtranscript, where it catalyzes the cleavage of the targeted transcript.It is also clear that all that is necessary to target a transcript fordegradation is that the sequence of the antisense strand of an siRNA becomplementary to that of a “target sequence” in the transcript to bedegraded. Empirical studies have shown, however, that not all targetsequences are equivalent. That is, siRNAs corresponding to differenttarget sequences within the same transcript can exhibit significantlydifferent efficiencies in directing the degradation of the sametranscript. Furthermore, at present it is impossible to predict whichtarget sequences will prove to be most effective targets. Practically,if one wants to efficiently silence a particular gene by RNAinterference, one must empirically determine which sequences make thebest targets by designing and testing siRNAs corresponding to differenttarget sequences. This process is both cumbersome and time consuming.

In all taxa exhibiting RNAi, siRNAs corresponding to a specific targetsequence in a gene or transgene (primary siRNAs or trigger siRNAs),evoke a “primary” RNAi response, wherein the targeted transcript iscleaved in the region of nucleotide sequence complementary to theantisense strand of the siRNA. However, in C. elegans and plants, a“secondary” RNAi response is observed, wherein “secondary” siRNAs areproduced that direct cleavage of the target transcript in regionsoutside of the original target sequence. In C. elegans, these secondarycleavages occur at sites exclusively 5′ of the primary siRNA targetsequence, but in plants, these secondary cleavages occur at sites either5′ or 3′ of the primary siRNA target sequence. Additionally, if theregions of nucleotide sequence in which secondary RNAi cleavages occurare homologous to other transcripts within the cell, the secondary RNAiresponse can lead to silencing of transcripts containing highly similarnucleotide sequences, that were not initially targeted by the triggersiRNA. The observed secondary RNAi response has been termed “transitiveRNAi,” because the sites of cleavage during the secondary responsetransit along the originally targeted transcript away from the primarytarget to adjacent regions, or the silencing transits to transcriptsthat were not initially targeted during the primary RNAi response.

Although the specific mechanisms operating behind the phenomenon oftransitive RNAi remain to be elucidated, the taxa that exhibittransitive RNAi also appear to be able to “amplify” the gene silencingresponse induced by primary siRNAs. Additionally, it has been shown thatin Arabadopsis and C. elegans, transitive RNAi requires the action ofputative RNA-dependent RNA polymerases (RdRPs) (Dalmay et al., Cell101:543-553 (2000) and Sijen et al., Cell 107:465-476 (2001)).Consequently, it has been hypothesized that in C. elegans, transitiveRNAi involves an amplification step catalyzed by RdRP, whereby theantisense strand of siRNAs serve as primers for synthesis of dsRNAs byRdRP, using the targeted transcript as the template, and the nascentdsRNAs are subsequently cleaved by an endonuclease (Dicer) to producesecondary siRNAs (Sijen et al., Cell 107:465-476 (2001). If the nascentdsRNA so synthesized contains nucleotide sequences highly similar tonucleotides sequences in RNA transcripts not targeted by the initialsiRNA during a primary RNAi response, the dsRNA corresponding to thesenucleotide sequences will be cleaved to form the “secondary” siRNAsnecessary to target these alternate transcripts and transit thesilencing to the gene products encoded by these alternate transcripts.This hypothesis is consistent with the observation that, in somestudies, siRNAs designed to target a particular member of a gene family,ultimately induced silencing of the entire family of genes.

Although RNAi has proven to be a remarkably powerful tool forinvestigating gene function in a variety of taxa, as mentioned above,Tuschl and colleagues only recently discovered that 21-nucleotide siRNAscould be used for studying gene function in mammalian cells withoutevoking a general antiviral response (Elbashir et al., Nature411:494-498 (2001)). Unfortunately, once a gene is selected forsiRNA-induced silencing, the choice of which sequences to target bysiRNAs is somewhat unclear. Towards this end, Holen and colleaguesinvestigated the efficacy of siRNAs targeted to different positions inthe transcript of human coagulation trigger Tissue Factor (hTF) in avariety of human cell types in culture (See Holen et al., Nucleic AcidsRes. 30:1757-1766 (2002)). In this study several siRNAs corresponding toseveral target sequences located in hTF transcripts were synthesized andtested for their ability to induce silencing of the hTF gene. Of theseveral siRNAs synthesized and tested only a few resulted in asignificant reduction in expression of hTF, suggesting that accessiblesiRNA target sites may be rare in some human mRNAs. Further, siRNAstargeting different sites in the hTF mRNA demonstrated strikingdifferences in their ability to silence the expression of hTF. Although,strong positional effects were seen with the siRNAs tested, and regionsof high GC content seem to be targeted less efficiently than those oflow GC content, Holen and coworkers concluded that the factorsdetermining the differences in siRNA efficiency remain unclear, and thatsusceptible RNAi target sites in some human genes may be rare.

From a practical perspective, the results of Holen and colleaguessuggest that it is difficult, if not impossible to predict, a priori,what sequences to target in a gene to target with siRNAs to induceefficient silencing by RNAi. In addition, there is a growing body ofevidence that specific siRNAs selected to silence particular genes mayproduce unwanted and unanticipated “off-target” effects—altering theexpression of untargeted RNA transcripts. Jackson and colleaguesrecently published the results of a study of off-target gene regulationconducted using a gene expression profiling technique to characterizethe specificity of gene silencing by siRNAs in cultured human cells.Their results provide clear evidence that treatment of cells with siRNAscorresponding to different sequences within the same RNA transcript mayresult in different, but reproducible, off-target silencing effects, atleast some of which may be due to partial sequence homology between theaffected transcripts and either the sense or the antisense strand of thesiRNA employed. They conclude that it may be difficult to select ansiRNA sequence that will be absolutely specific for the target ofinterest (Jackson et al., Nature Biotech. 21:635-637 (2003)).

Recent advances in genomics, especially with the completion of the humangenome sequence, have lead to the discovery of numerous novel genes ofunknown function. Unfortunately, advances in our ability to sequencegenomes, and identify novel genes within them have far outstripped ourability to determine the function of the gene products of these novelgenes. Classically, gene function has been addressed in vivo by twodistinct approaches: overexpression of the gene product andunderexpression of the gene product.

High-throughput methodology in biotechnology is largely responsible forthe recent explosive growth of knowledge in genomics and proteomics—twospecialty fields that are relatively new to the larger field ofmolecular biology. In the realm of pharmaceutical research anddevelopment, high-throughput technologies, genomics and proteomics, havehad a profound impact on therapeutic drug development (Kennedy. EXS.89:1-10 (2000)). Such technologies have issued in a “new millennium” ofdrug discovery (Cunningham. J. Pharmacol. Toxicol. Methods. 44:291-300(2000)), and have provided a catalyst for change in drug discoveryparadigms (Hanke. J. Law Med. Ethics. 28(4 Suppl):15-22 (2000)). Suchtechnologies have the potential for greatly increasing the speed of drugdevelopment, and for reducing the associated costs—both of these factorsbeing critically important given the current economic and socialclimate. Clearly, significant improvements in the ability of researchscientists to (a) selectively overexpress specific target genes, (b)selectively block the expression of specific target genes, (c) screenlarge numbers of target genes for their cellular functions, and (d)ultimately determine how overexpression or underexpression of specifictarget genes affect desired outcomes in mammalian cells, will be ofbenefit to society.

Traditionally, RNAi has been applied to investigate the function ofgenes in a one-target-at-a-time mode. This approach has proven veryuseful in analyzing the function of a limited number of genes in themodel organisms C. elegans and D. melanogaster. The use ofmicroarray-based RNAi technology using siRNAs should greatly facilitatethe investigation of functions for hundreds or thousands of mammaliangenes simultaneously in a parallel fashion. However, given the findingsof Holen et al. and Jackson et al., discussed above, the choice of whatspecific sequence within an mRNA to target with siRNAs of correspondingsequence is completely unclear. Further, although microarray-based RNAitechnology will perhaps allow the empirical identification of sequencesthat will serve as ideal targets of RNAi, the process of synthesizingand testing numerous siRNAs is laborious and costly.

While RNAi holds much promise for high-throughput analyses designed todetermine gene function through the silencing of large numbers of genes,the method is not without complications and challenges. Perhaps the mostfundamental challenge is how to pick a target sequence within the targettranscript that will allow for the efficient silencing of the targetgene, along with minimal unintended and undesired off-target effects.Despite numerous studies in which siRNAs have been employed to inducegene silencing, no definitive rules have evolved to assist researchersin picking the most effective sequences to target within a giventranscript. Although there are general guidelines to help researchersnarrow their choices for target sequences, researchers must still use atrial and error approach to empirically determine what individual siRNAswork best, and what siRNAs have minimal off-target effects. Given theselimitations and the many potential and varied applications of RNAi,there is a clear need for alternative approaches and techniques foraltering gene expression by siRNAs, especially with regards tohigh-throughput applications

BRIEF SUMMARY OF THE INVENTION

The present invention provides novel methods for altering levels ofexpression of a plurality of gene products using RNA interference (RNAi)induced by a “Universal interfering RNA” (UiRNA) directed towards acommonly-shared “Universal target RNA” (UtRNA). The UtRNA, which isincorporated into a plurality of chimeric RNA transcripts, eachcomprising a different subject RNA, whose cellular concentration is tobe decreased, may be located at several locations within the chimerictranscripts, depending upon the application. In addition, when thesubject RNAs encode polypeptides, a UtRNA encoding a readily detectablepeptide (e.g., epitope tag, fluorescent peptide, enzymatic tag, etc.)can be chosen and cloned in-frame with the subject RNAs such that fusionproteins are produced by translation of the chimeric RNA transcripts.Advantageously, such a UtRNA-encoded peptide tag can be used to readilyquantitate the level of expression of the fusion proteins under study.The methods of the present invention can be readily employed toinvestigate the function of any number of gene products, and the moregene products being investigated, the greater the benefits of themethods. Preferably the method is employed to selectively manipulate theexpression levels of a plurality of distinct gene products in culturedtarget cells, in a parallel, high-throughput, format.

Importantly, the expression of all gene products encoded by chimeric RNAtranscripts bearing a common UtRNA can be manipulated through RNAiinduced by the same UiRNA, which is specifically designed to target theUtRNA. As such, the UiRNA can be a double-stranded siRNA, or asingle-stranded shRNA. Furthermore, the UiRNA can be introduced into thetarget cells by any of the various means known in the art. In one set ofembodiments, introduction of the UiRNA is by way of a DNA that directsthe in vivo transcription of an RNA, or RNAs within the target cells ororganisms. In certain embodiments the in vivo transcribed RNA can be asmall hairpin RNA (shRNA) that is processed by cellular ribonucleases(RNases) to produce an siRNA-like UiRNA. In other embodiments, twocomplementary siRNAs can be transcribed in-vivo, and annealing of thetwo individual strands results in the formation of a double-strandedsiRNA-like UiRNA. In some of these embodiments the DNA that directs thein-vivo expression of the UiRNA is introduced into the target cells ororganisms before the introduction of expression vectors directing theexpression of the chimeric RNA transcripts. In certain of theseembodiments, an expression cassette—and preferably an inducibleexpression cassette—is stably introduced into the target cells ororganisms to produce transgenic target cells or organisms that canexpress UiRNA when required to do so. In another set of embodiments, theUiRNA is synthesized in vitro and subsequently introduced to the targetcells. Such in vitro synthesized UiRNA may be chemically orenzymatically synthesized and may be the double-stranded UiRNA itself,or may be an shRNA that is processed by RNases, in vivo or in vitro, toproduce the UiRNA.

The UtRNA can be composed of any sequence of nucleotides thatfacilitates the efficient RNAi of all chimeric RNA transcripts in whichit appears, regardless of where it occurs within the transcript, andregardless of what target RNA is included in the same transcript. In oneset of embodiments, the UtRNA is placed in a non-coding region of thechimeric RNA transcript—either the 5′ untranslated region (UTR), or the3′ UTR. In another set of embodiments, the UtRNA encodes a peptide andis inserted in-frame with the coding region of the target RNA—eitherin-frame with either end of the coding region, or within the codingregion itself—thereby creating a chimeric open reading frame that istranslated into a fusion protein. In this set of embodiments, the UtRNApreferably encodes a peptide, which can be used to detect and quantitatethe level of expression of the fusion protein.

Advantageously, the methods of the present invention provide a means fortargeting a plurality of recombinantly expressed gene products for RNAinterference, by providing a plurality of expression vectors that directthe expression of a plurality of chimeric RNA transcripts, eachcomprising a different, unique subject RNA preferably encoding aparticular gene product, and a common UtRNA. These expression vectorsare then introduced into target cells or organisms, which are capable oftranscribing the chimeric RNA transcripts and translating the encodedgene products, thereby creating a plurality of transfected target cellsor organisms, each collection of target cells or organism transfectedwith the same expression vector expressing the same chimeric RNAtranscript and encoded gene product. In a preferred embodiment thetransfected target cells or organisms are arranged in an addressablearray, with cells or organisms transfected with a single expressionvector exclusively occupying a specific address within the array. AUiRNA that is capable of inducing RNAi by targeting the common UtRNAshared by all expression vectors and chimeric RNA transcripts, in alltransfected target cells or organisms can then be introduced into thesetransfected target cells or organisms to simultaneously reduceexpression of all recombinantly expressed gene products. As mentionedabove, the means by which the UiRNA is introduced into the transfectedtarget cells include introduction by way of a DNA that directs the invivo transcription of RNA, as well as by introduction of in vitrosynthesized RNA. Also, as mentioned above, the RNA transcribed orsynthesized can be a double-stranded siRNA-like UiRNA, or can be a shRNAthat is processed by cellular RNases to form the UiRNA.

The methods of the present invention also provide a means ofinvestigating the effects of altered levels of gene product expressionfor a plurality of gene products by providing a plurality of expressionvectors that are introduced into a cell and direct the expression of achimeric RNA transcripts, each encoding a particular gene product andbearing a common UtRNA. These expression cassettes are introduced intotarget cells that are capable of transcribing and translating thechimeric RNA transcripts; thereby creating a plurality of transfectedtarget cells that overexpress the gene product cloned into theexpression cassette (also referred to as the gene product under study).At some point in time relative to the introduction of the expressioncassette, UiRNA is introduced into the transfected target cells. Theintroduced UiRNA, which corresponds in sequence to the UtRNA, inducesRNAi and promotes the degradation of the chimeric RNA transcriptsbearing the UtRNA. The net result is a reduction in the expression ofthe particular gene product expressed from the chimeric RNA transcriptproduced in that cell. Differences in the transfected target cellsbefore and after introduction of the UiRNA are detected and measured,and are preferably correlated with differences in the levels ofexpression of the gene product. Using such an approach the effects ofaltered gene expression are observed, and the function of the geneproduct whose expression levels are manipulated is revealed.

Importantly, the methods of the present invention can be employed tomanipulate levels of expression of gene products in cells, tissues, ororganisms transfected with the expression cassettes of the presentinvention. In particular, the type of RNA introduced to induce RNAi, theamount of RNA introduced (dosage), the means by which it its introduced(route and method of introduction—e.g., lipofection, electroporation orsynthesis in vivo) can be adjusted to produce the quantitative andtemporal manipulation of gene product expression desired.

The methods of the present invention can be used with any type of cellthat (1) can be transfected with the expression cassettes of the presentinvention, (2) will direct the production of the chimeric RNAtranscripts of the present invention, (3) to which UiRNA can beintroduced, and (4) that exhibits RNAi. Importantly, the methods of thepresent invention can be used in a variety of cell types or cell lines,and different cell types or cell lines can be chosen to create differentexperimental scenarios. In particular, cell lines that show distinctlydifferent endogenous levels of expression of the gene product understudy, or no expression of the gene product under study, can be chosento augment the methods of the present invention for studying geneproduct function. Additionally, the methods of the present invention canbe employed in cell types, cell lines, tissues or organisms that exhibittransitive RNAi, with very different effect. These different scenariosare presented in more detail below.

The present invention further provides kits that can be used to eithertarget a plurality of recombinantly expressed gene products for RNAinterference, or to determine the effects of altered levels of geneproduct expression. These kits can comprise one or more vectors,including expression vectors having an expression cassette comprising amultiple cloning site and a UtRNA, wherein the expression cassette iscapable of directing the expression of a recombinant transcriptcomprising the UtRNA and any nucleotide sequence inserted into themultiple cloning site (a subject RNA). The kits further comprise eitherinterfering RNAs that effectively induce the RNAi of expression of therecombinantly expressed gene product, or transcription vectors thatdirect the in vivo transcription of such RNAs. These RNAs, which aredesigned to induce RNAi by specifically targeting the UtRNA contained inthe recombinant transcripts, correspond in sequence to the UtRNA.

In one set of embodiments, the present invention provides arrays ofcultured cells, tissues, or organisms wherein the cells, tissues, ororganisms at specific addresses in the array have been transfected withexpression vectors comprising a chimeric expression cassette having anucleotide sequence encoding a particular gene product, and a commonUtRNA. The chimeric expression cassettes in these transfected andarrayed cultures of cells, tissues or organisms are capable of directingthe expression of chimeric RNA transcripts encoding different geneproducts, but bearing the same UtRNA. Consequently, the expression ofall gene products in all transfected and arrayed cultures of cells ororganisms can be subjected to a reduction of expression by inducing RNAiwith a common UiRNA, that is administered to the cells or organisms.Such arrays of transfected cultured cells should be made up of at least2, 3, 4, 5, preferably 6, 8 or 12, and more preferably 16, 24, 32, 36,40, 48, 56, 60, 64, 72, 80, 84, 88, 96 or more groups of cells ororganisms transfected with different chimeric expression vectors, allcapable of directing the expression of chimeric RNA transcripts encodingdifferent gene products, but bearing a common UtRNA. Such arrays oftransfected cultured cells, tissues, or organisms can be arranged instrips of 2, 3, 4, 6, 8, 12, or more culture tubes or vessels, or in96-well plates, or the likes. Cells in such arrays can be grown insuspension, or as a monolayer on a substrate. Alternatively, microarraysof transfected cells can be made in a monolayer of cells attached to acommon substrate as described by Sabatini in U.S. Pat. No. 6,544,790,which is incorporated by reference herein in its entirety.

In another set of embodiments, the present invention further providesmethods by which the involvement and/or role of a plurality of geneproducts in pathogenesis, genetic disorders, or infectious diseases canbe assessed. For these embodiments, the nucleotide sequences encodinggene products suspected or known to be associated with or involved in aparticular infectious disease or genetic disorder are inserted into theexpression cassettes of the present invention. These expressioncassettes, which may be further incorporated into expression vectors,are introduced into cells of an appropriate cell type or line to producetransfected target cells or tissue, or into whole organisms to producetransfected organisms. Once inside the cells or organisms, theexpression cassettes direct the production of chimeric RNA transcriptsthat encode the different suspect gene products, but bear a commonUtRNA. The levels of expression of the encoded gene products can befurther manipulated in the transfected target cells or organisms by theintroduction of RNA(s) corresponding in sequence to the UtRNA thatinduce RNAi by targeting the UtRNA (UiRNAs). The transfected targetcells or organisms can then be examined in the presence and absence ofsuch UiRNAs, to determine whether the gene product whose expressionlevel is being manipulated by RNAi, does indeed play a role inpathogenesis, genetic disorders, or infectious diseases. For example,the transfected target cells or organisms can be exposed to aninfectious agent in the absence of UiRNA (conditions under whichexpression of the gene product encoded in the chimeric RNA transcriptswill be enhanced), or in the presence of UiRNA (conditions under whichexpression of the gene product encoded in the chimeric RNA transcriptswill be silenced or reduced). If the gene product under study plays arole in the infectious cycle of the infectious agent, differences in thehealth of the transfected target cells or organisms will likely beobserved between identical cultures of the transfected target cells ororganisms maintained under the two different treatment regimes (i.e., inthe presence or absence of UiRNA).

In still another set of embodiments, the present invention also providesmethods of treating disease by manipulating the levels of expression ofa particular gene product. Such methods may be applied to human cells,tissues or patients, or may be applied to non-human cells, tissues ororganisms. For humans, the method comprises the steps of administeringto a person in need of such treatment a chimeric expression cassettecomprising a nucleotide sequence that directs the expression of achimeric RNA transcript encoding a particular gene product and a UtRNA,and introducing into the person an RNA that induces RNAi of expressionof the encoded gene product by targeting the UtRNA in the recombinanttranscript. As above, the step of introducing the siRNA can be by way ofintroducing a DNA that directs the in vivo transcription of an RNA thatinduces RNAi, or alternatively, can be by way of introducing an RNA thathas been synthesized in vitro and induces RNAi in vivo. The RNAintroduced can be a double-stranded siRNA, or a single-stranded hairpinRNA that is processed into an siRNA by RNases. RNAs that are synthesizedin vitro can be synthesized either enzymatically or chemically. The RNAcan include modified nucleotides of any type, as well as other chemicalmodifications that impart characteristics such as improved stability,resistance to nucleases, greater efficacy, etc.

The diseases being treated in human can be genetic disorders orinfectious diseases. The infectious diseases can be diseases caused byany type of pathogen, including viruses, bacteria, fungi, parasiticprotozoa, nematodes, etc. Viral diseases that can be treated includediseases caused by human immunodeficiency virus (HIV), hepatitis B virus(HBV), hepatitis C virus (HCV), human herpesvirus 1 (HSV1), humanherpesvirus 2 (HSV2), human influenza A (HIA), and the like. Also, thegene products whose expression levels are being manipulated can bemammalian gene products, human gene products or pathogen gene products,including viral gene products.

Advantageously, in cells or organisms that naturally exhibit, or aremade to exhibit, transitive RNAi, the methods of the present inventionfurther provide a means to reduce the expression of recombinantlyexpressed gene products, as well as endogenous gene products. In suchorganisms, or the cells from such organisms, RNAi induced gene silencingcaused by UiRNAs that target UtRNAs in expressed chimeric RNAtranscripts would likely transit or propagate to transcripts bearingsequences either identical to, or highly similar to the sequence encodedin the chimeric transcripts. Hence, in such cells or organisms themethods of the present invention can be used to promote the silencing ofexpression of any endogenous gene product without having to design oradminister gene-specific siRNAs. Further, multiple endogenous geneproducts can potentially be silenced in such organisms or cells using asingle UiRNA that is targeted to the UtRNA in a plurality of chimericRNA transcripts that also bear the sequences encoding the endogenousgene products to be silenced (subject RNAs).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts five different exemplary configurations of chimeric RNAtranscripts that can be produced from five different types of expressioncassettes of the present invention. Each of the five configurationsincludes a 5′ UTR, 3′ UTR, nucleotide sequence that encodes the geneproduct whose expression is to be manipulated (the subject RNA), and auniversal target RNA (UtRNA). In the first two examples the UtRNA isfound in non-coding sequence, and the last three examples the UtRNAencodes a peptide, preferably a readily detectable peptide, and iscloned in-frame with the open reading frame encoding a particular geneproduct;

FIG. 2 depicts how the methods of the present invention can be used inan array-based format to target a plurality of gene products for RNAiwith a single type of UiRNA, thereby simultaneously manipulating theexpression levels of this plurality of gene products;

FIG. 3 illustrates how a UiRNA is used to target a chimeric RNAtranscript for degradation in target cells expressing a correspondingendogenous transcript, wherein the target cells do not exhibittransitive RNAi;

FIG. 4 illustrates how a UiRNA is used to target a chimeric RNAtranscript for degradation in target cells that do not express acorresponding endogenous transcript, and wherein the target cells do notexhibit transitive RNAi; and

FIG. 5 illustrates how the methods of the present invention can be usedto manipulate gene expression in target cells that express a chimericRNA transcript, as well as a corresponding endogenous transcript, andalso exhibit transitive RNAi.

DETAILED DESCRIPTION OF THE INVENTION 1. Definitions

As used herein, the term “RNA interference,” or “RNAi,” refers to theprocess whereby sequence-specific, post-transcriptional gene silencingis initiated by an RNA that is homologous in sequence to the silencedgene. RNAi, which occurs in a wide variety of living organism and theircells, from plants to humans, has also been referred to aspost-transcriptional gene silencing (PTGS) and co-suppression indifferent biological systems. The sequence-specific degradation of mRNAobserved in RNAi, is mediated by small (or short) interfering RNAs(siRNAs).

As used herein, the term “transitive RNA interference,” refers to theprocess whereby RNAi induced by an administered or introduced siRNA (theprimary siRNA) leads to the in vivo formation of a population ofsecondary siRNAs, generally corresponding to nucleotide sequencesnearby, but distinct from, the nucleotide sequence targeted by theprimary siRNA. These secondary siRNAs induce a secondary RNAi responsein which transcripts bearing nucleotide sequences corresponding to thesecondary siRNAs are targeted for endonucleolytic cleavage. The term“transitive RNAi” refers to the ability of secondary siRNAs to “transit”the RNAi gene-silencing phenomenon from the originally targetedtranscript to different transcripts bearing regions of nucleotidesequence homologous to the sequences of the secondary siRNAs.

As used herein, the term “interfering RNA” means an RNA molecule capableof directing the degradation of an RNA transcript having a nucleotidesequence at least a portion of which is substantially the same as thatof the interfering RNA, through the mechanism of RNA interference(RNAi). As known in the art, interfering RNAs can be “small interferingRNAs,” or siRNAs, composed of two complementary single-stranded RNAsthat form an intermolecular duplex. Interfering RNAs can also be “shorthairpin RNAs,” or shRNAs, composed of a single-stranded RNA with twoself-complementary regions that allow the RNA to fold back upon itselfand form a stem-loop structure with an intramolecular duplex region andan unpaired loop region. Finally, in some circumstances (See Martinez etal., Cell 110:563-574 (2002)), interfering RNAs can be single-strandedantisense RNAs of 19 to 29 nucleotides that are complementary to atarget sequence.

The terms “small interfering RNA” (also sometimes referred to as shortinterfering RNA) or “siRNA,” as used herein, refer to the mediators ofRNAi-RNA molecules capable of directing sequence-specific,post-transcriptional gene silencing of specific genes with which theyshare nucleotide sequence identity or similarity. Recent experimentshave shown that the siRNAs that are most effective in mammalian cellsare duplexes composed or two complementary 21 nucleotide single-strandedRNAs that anneal to form a duplexed region of 19 basepairs andsingle-stranded overhangs of 2 nucleotides at their 3′ ends. In someorganisms (e.g., C. elegans, D. melanogaster and various plants) thesesiRNAs can be created by the nucleolytic processing of longer dsRNAs. Inmammalian cells they apparently can also be produced from short (i.e.,less than 30 basepairs) hairpin RNAs, or shRNAs.

The term “small hairpin siRNA,” “short hairpin siRNA,” or “shRNAs,” asused herein, refers to siRNAs composed of a single strand of RNA thatpossesses regions of self-complementarity that cause the single strandto fold back upon itself and form a hairpin-like structure with anintramolecular duplexed region containing at least 19 basepairs.Importantly, because they are single-stranded, shRNAs can be readilyexpressed from single expression cassettes.

The term “knock down,” as used herein, describes the condition createdby RNAi, wherein the expression of a particular gene-product, or thecellular concentration of a particular RNA transcript, is reduced oreliminated by the sequence-specific, post-transcriptional gene silencinginitiated by siRNAs that are homologous in sequence to the gene encodingsaid gene product.

The term “subject RNA,” as used herein, refers to an RNA whose cellularconcentration is to be altered, manipulated or reduced, or knocked down,by the action of an interfering RNA targeting the universal target RNA,but not the subject RNA.

As used herein, the term “chimeric RNA transcript” means an RNAtranscript comprising a subject RNA operably linked to a universaltarget RNA to create a single RNA that does not naturally occur innature.

The term “operably linked,” when used in the context of a chimeric RNAtranscript, means joined directly or indirectly such that the universaltarget RNA facilitates the reduction in concentration of at least thesubject RNA when the chimeric RNA transcript is subjected to RNAinterference induced by a universal interfering RNA.

The term “universal target RNA,” or UtRNA, as used herein, refers to acommon RNA that is incorporated into a plurality of chimeric RNAtranscripts, and serves to impart upon the chimeric RNA transcripts asusceptibility to degradation by RNA interference promoted by a“universal interfering RNA” targeting the universal target RNA.

The term “in vitro transcription,” as used herein, describes the processwhereby a new (nascent) RNA molecule is synthesized, outside of a livingcell, from individual ribonucleotides-triphosphates by an enzyme(usually RNA polymerase), using a DNA molecule as a template to specifythe sequence of the nascent RNA.

As used herein, the term “transcription vector” refers to a vectorcontaining, at least, a promoter that directs transcription by an RNApolymerase, a transcription template sequence, and a transcriptionterminator sequence, such that an RNA transcript can be synthesized fromthe transcription vector.

2. Overview and Embodiments

The present invention describes a novel method for manipulating theexpression of a plurality of gene products within a cell or organismusing a UiRNA that is designed to specifically target a UtRNA. Themethods of the present invention, which can be employed in numerousalternative embodiments, can be used for a variety of purposes.Specifically, the methods of the present invention can be used to targeta plurality of recombinantly expressed gene products for RNAi using asingle, common UiRNA. The methods of the present invention can also beused to determine the effects of altered levels of gene expression,preferably for a plurality of genes, and preferably in arrayedcollections of cells. The methods of the present invention can also beused to identify gene products that play important roles inpathogenesis, genetic disorders, and infectious diseases.

The present invention also provides kits that can be used to target aplurality of recombinantly expressed gene products for RNA interferenceusing a single, common UiRNA. The present invention further provides forarrays of transfected cells, preferably created through the use oftransfection microarrays, in which a single UiRNA can be used to induceRNAi of a plurality of gene products, each expressed in a collection ofcells at a specific address within the array. The methods of the presentinvention, when utilized in organisms or cells exhibiting transitiveRNAi, provide a method of reducing the expression of a plurality ofendogenous genes, using a UiRNA that is targeted to a UtRNA, when anucleotide sequence encoding the endogenous gene, or a homologuethereof, is included along with a UtRNA in a single chimeric RNAtranscript. Consequently, in organisms or cells that either naturallyexhibit transitive RNAi, or are made to carry out transitive RNAi, themethods of the present invention provide a method for treating diseasethat involves reducing the cellular concentration of gene productsencoded by endogenous RNA transcripts by first targeting a chimeric RNAtranscript bearing a UtRNA and encoding a gene product whose endogenousexpression is desired to be reduced, using UiRNA-induced RNAi toinitially reduce the expression of the recombinantly expressed geneproduct, and allowing secondary siRNAs generated during that process totransit and cause the RNAi-mediated degradation of endogenoustranscripts encoding the target gene product, and homologues thereof.These various embodiments are described in detail below.

It should be stressed that the methods of the present invention can beexpected to have very different effects, and hence different uses, incells and organisms that naturally exhibit transitive RNAi, or can bemade to do so, as opposed to those that do not. In the former—cells andorganisms that naturally exhibit transitive RNAi, or can be made to doso—the methods of the present invention can be expected to result in anet reduction of expression of the plurality of gene products encoded inthe plurality of expression vectors tested. This net reduction ofexpression results from the production of secondary siRNAs, which cantransit to target homologous endogenous transcripts and promote theirendonucleolytic cleavage. In such cells and organisms, the methods ofthe present invention can be expected to reduce the expression of thegene products under study to levels below those naturally occurring inuntreated target cells. In contrast, in cells or organisms that do notexhibit transitive RNAi, the methods of the present invention can beexpected to result in levels of expression of the plurality of geneproducts encoded in the plurality of expression vectors tested somewherebetween those of untransfected target cells (i.e., the naturalbackground levels), and cells transfected with the expression vectors ofthe present invention (i.e., the overexpressed levels). In these cellsand organisms, the methods of the present invention can be used toinvestigate the effects of overexpression of the plurality of geneproducts encoded in the plurality of expression vectors tested, followedby a partial reduction in their expression level. In such cells andorganisms the methods of the present invention can be useful forinvestigating the disregulation of expression of the plurality of geneproducts encoded in the plurality of expression vectors tested.

The present invention provides a method for manipulating the levels ofexpression of a plurality of gene products using a UiRNA that istargeted to a UtRNA incorporated into chimeric RNA transcripts expressedfrom expression cassettes or expression vectors in target cells. Thesechimeric RNA transcripts, which can encode any gene product, comprise asubject RNA and a UtRNA, and the UtRNA can be incorporated into thechimeric RNA transcripts in any location with respect to the subject RNA(FIG. 1). The method overcomes the limitations faced in the prior art.First, the same UtRNA is incorporated into a plurality of chimeric RNAtranscripts, a single type of UiRNA, which targets that that UtRNA, canbe used to reduce the expression of all gene products encoded by thatplurality of chimeric RNA transcripts. Second, the UtRNA targeted by theUiRNA, and the UiRNA employed, can be selected and tested in advance, inorder to assure that (a) UiRNA-mediated, RNAi-induced, gene silencingworks effectively and without regard to the location of the UtRNA in thechimeric RNA transcripts, or the identity of the gene product codingsequence included within the chimeric RNA transcripts, and (b) the UiRNAemployed results in minimal, or negligible off-target gene silencing.These advantages are discussed in further detail below.

Currently, in order for researchers to use RNAi to effectively silencethe expression of specific heterologous recombinant or endogenous geneproducts, they must design and test specific siRNAs that target that thetranscript encoding the particular gene product whose levels ofexpression they wish to reduce. Each gene to be silenced requires that aspecific siRNA, or set of siRNAs, be designed to promote RNAi-inducedsilencing, and each siRNA designed must be tested for its efficacy.Since not all sequences within transcripts encoding particular geneproducts are efficiently targeted by corresponding siRNAs, researchersmust generally conduct preliminary experiments to empirically determinewhich sequences, and their corresponding siRNAs, work best forRNAi-mediated gene silencing (see Holen et al., Nucleic Acids Res.30:1757-1766 (2002)).

Similarly, as described above, there is a growing body of evidence thatspecific siRNAs selected to silence particular genes may produceunwanted and unanticipated “off-target” effects—altering the expressionof untargeted RNA transcripts. In fact, treatment of cells with siRNAscorresponding to different sequences within the same targeted RNAtranscript has been shown by expression profiling to result indifferent, but reproducible, off-target silencing effects. Apparently,at least some of these off-target effects may be due to partial sequencehomology between the affected transcripts and either the sense or theantisense strand of the siRNA employed, but some could not be explainedby sequence homology, leading the researchers to conclude that it may bedifficult to select an siRNA sequence that will be absolutely specificfor the target of interest (Jackson et al., Nature Biotech. 21:635-637(2003)). Since the UtRNA of the present invention, and the UiRNA whichtargets it, can be selected and tested in advance, such undesirableoff-target effects can be minimized. Furthermore, since the chimeric RNAtranscripts of the present invention that encode different gene productsin the subject RNA bear the same UtRNA, they can all be silenced byintroduction of the same UiRNA. And since Jackson and colleagues haveshown that the off-target silencing effects of specific siRNAs aredistinct and reproducible, use of the same UiRNA for the induction ofRNAi in different target cells bearing different chimeric RNAtranscripts, at least results in equivalent off-target silencing effectsin all target cells, thereby eliminating any variability that mightotherwise result from the introduction of different,transcript-specific, siRNAs to different target cells.

Certainly, incorporating a common UtRNA in a plurality of chimeric RNAtranscripts, and targeting those chimeric RNA transcripts forRNAi-induced degradation through the introduction of a singlecorresponding UiRNA, eliminates the need for designing and testingdifferent gene-specific siRNAs and may reduce or eliminate thevariability of silencing effects caused by introduction of differentgene-specific siRNAs. Further, a UtRNA and its corresponding UiRNA canbe chosen and tested in advance to ensure that (a) the expression ofgene products encoded by recombinant transcripts bearing a UtRNA areeffectively silenced by introduction of a corresponding UiRNA, and (b)the silencing observed is specific. Additionally, and advantageously, aUtRNA that functions effectively and specifically in one chimeric RNAtranscript, should function equally well in other chimeric RNAtranscripts encoding different gene products. Similarly, since UtRNAscan be identified that have little or no similarity to any endogenousnucleotide sequences, UiRNAs targeting these UtRNAs can be expected tohave little or no homology-related effect on endogenous geneexpression—unless the cell or organism in which the UtRNA/UiRNA systemis employed exhibits transitive RNAi. In which case, UiRNA-inducedsilencing of expression of a chimeric transcript having a subject RNAoperably linked to a UtRNA may be expected to “transit” to endogenoussequences, leading to their silencing as well. In such cells ororganisms, use of pre-selected UiRNA targeted to a UtRNA in a chimericRNA transcript should lead to the silencing of corresponding endogenousgenes or gene families.

Since a single UtRNA can be incorporated into any number of chimeric RNAtranscripts, and that UtRNA can be targeted by a single type of UiRNA,another significant advantage of the present invention is that themethods disclosed can be used to effectively manipulate the expressionof a plurality of gene products. Such manipulation of expression of aplurality of gene products may be done either simultaneously ornonsimultaneously. Consequently, the methods of the present inventioncan be used to determine the effects of altered levels of expression ofa plurality of gene products in a plurality of cells, or cell clustersor cultures, or organisms transfected with expression cassettesdirecting the expression of chimeric RNA transcripts bearing a commonUtRNA, but encoding different gene products. Specifically, groups oftransfected cells or organisms can be prepared, each group transfectedwith a different expression cassette that directs the expression of aspecific chimeric RNA transcript encoding a unique gene product in asubject RNA, but bearing a common UtRNA. These groups of cells ororganisms, each of which directs the overexpression of the particulargene product encoded by their expression cassette, can be treated withthe same UiRNA to reduce the expression of these particular geneproducts (FIG. 2). Consequently, the method of the present inventionprovide a means for determining the effects of altered levels ofexpression of a plurality of gene products, using a single type of siRNAto promote RNAi-induced silencing of expression of the heterologous geneproducts encoded in the various chimeric RNA transcripts. Such groups oftransfected cells or organisms can be cultured, split into a pair ofcultures and one member of the pair can be treated with UiRNA. Cells ororganisms from the treated and untreated paired cultures can then becompared to determine the effects of altered levels of expression of theparticular gene product encoded by the expression cassette carried bythose cells or organisms. The comparison of treated and untreated pairedcultures can be by any means known to the art. Comparisons could, forexample, involve comparing growth rates, comparing cellularmorphologies, comparing the expression of specific cellular markers, orcomparing the performance or behavior of the cells or organisms inspecific diagnostic assays.

In preferred embodiments of the present invention the UtRNA employed canbe designed to encode a readily detectable peptide, such as an epitopetag, fluorescent peptide, enzymatic tag, etc., such that, when clonedin-frame with a nucleotide sequence encoding a gene product whoseexpression is to be manipulated, the UtRNA directs the addition of thedetectable peptide to the expressed gene product. The UtRNA encoding thedetectable peptide can be added to either end of the sequence encodingthe gene product to be manipulated so that the detectable peptide tag isadded to one end or the other of the native gene product (FIG. 1).Alternatively, the UtRNA can be cloned within the sequence encoding thegene product to be manipulated, so that the detectable peptide tag isfound imbedded within the natural polypeptide sequence of the geneproduct (FIG. 1). Preferably, and regardless of the location chosen forthe detectable peptide tag encoded by the UtRNA and added to thepolypeptide sequence of the gene product whose expression is to bemanipulated, the detectable peptide tag added to the expressed geneproduct facilitates the ready detection, quantitation, and perhapslocalization of the resulting expressed fusion protein. Hence, thedetectable peptide tag can be exploited by researchers to track orfollow not only the levels of expression of the recombinantly expressedfusion protein directed by the expression cassette in which it isencoded, but also can be used to track or follow the effects oftreatment of such cells with corresponding UiRNAs, or control siRNAs. Inother words, the detectable peptide tag present on a recombinantlyexpressed fusion protein allows researchers to readily assess whetherthe desired gene is being overexpressed at the protein level intransfected target cells before the introduction of UiRNA, and whetherthe expression of the protein is diminished or silenced following theintroduction of, or treatment with, siRNAs.

On the other hand, the UtRNA need not necessarily be designed to encodea detectable peptide, and need not necessarily be cloned within, ordirectly adjacent to the coding region encoding the gene product whoseexpression is to be manipulated. The expression cassettes of the presentinvention may be designed and assembled such that the UtRNA ends up inthe 5′ untranslated region, or in the 3′ untranslated region of therecombinant transcript encoding a gene whose expression is to bemanipulated (FIG. 1). In essence, the location of UtRNA in the chimericRNA transcript is a matter of choice, so long as the UtRNA allows forthe effective targeting of the chimeric RNA transcript for RNAi-induceddegradation of the subject RNA when cells (or organisms) are treatedwith UiRNAs of corresponding sequence.

Importantly, cell types or cell lines vary with respect to their basallevels of expression of a particular gene product or family of geneproducts. Clearly, the type or line of cells chosen for practicing themethods of the present invention will have a profound effect on theresults one will attain using the methods of the present invention,since different cell types or cell lines exhibiting different basallevels of endogenous expression of various gene products can be employedto practice the methods of the present invention. Generally speaking,and for the purpose of practicing the methods of the present invention,all cell types can be grouped into one of two categories, based onwhether the cells of that cell type or line express a particular geneproduct or not. All cell types can be further divided into one of twocategories based on whether they exhibit transitive RNAi or not. Asdescribed above, transitive RNAi refers to the process whereby RNAiinduced by an administered siRNA (the primary siRNA) leads to thesecondary silencing of non-targeted transcripts, such as transcripts ofhomologous sequence. As will be seen, the methods of the presentinvention can be expected to have significantly different net effectswith respect to the manipulation of expression of a particular geneproduct depending upon in which of the resulting four categories thecell type or line falls (i.e., (a) expressing a particular gene productfrom an endogenous gene, and not exhibiting transitive RNAi, (b) notexpressing a particular gene product from an endogenous gene, and notexhibiting transitive RNAi, (c) expressing a particular gene productfrom an endogenous gene, and exhibiting transitive RNAi, and (d) notexpressing a particular gene product from an endogenous gene, andexhibiting transitive RNAi). While not wishing to be bound by anytheory, the methods of the present invention are particularly useful incells and organisms that either naturally exhibit transitive RNAi, orare otherwise made to exhibit transitive RNAi. These four categorieswill be discussed separately below.

In the first category (FIG. 3), before transfection with an expressionvector of the present invention, the cell type or cell line expresses aparticular gene product (protein P_(E)) from an endogenous gene, but thecell does not exhibit transitive RNAi. Following transfection with anexpression vector of the present invention, the transfected cells of thecell type or cell line express the gene product from both the endogenousgene (protein P_(E)), and from the chimeric RNA transcripts of thepresent invention (protein P_(R)*). Following introduction of the UiRNA,which targets the UtRNA of the chimeric RNA transcript, expression ofthe gene product from the chimeric RNA transcripts of the presentinvention (protein P_(R)*) is silenced, but expression of the geneproduct from the endogenous gene (protein P_(E)) continues. In thiscategory, following the introduction of the UiRNA, the levels ofexpression of the gene product under study are not likely to drop belowthe initial level resulting from basal expression from the endogenousgene.

In the second category (FIG. 4), before transfection with an expressionvector of the present invention, the cell type or cell line does notexpress a particular gene product (protein P_(E)) from an endogenousgene, and the cell does not exhibit transitive RNAi. Followingtransfection with an expression vector of the present invention, thetransfected cells of the cell type or cell line express the particulargene product from the chimeric RNA transcripts of the present invention(protein P_(R)*). Following introduction of the UiRNA, which targets theUtRNA of the chimeric RNA transcript, expression of the gene productfrom the chimeric RNA transcripts of the present invention (proteinP_(R)*) is silenced. In this category, following the introduction of theUiRNA, the levels of expression of the gene product under study willgenerally drop significantly below the maximum levels of expressiondirected by the transgene, but the amount by which the expression levelis reduced will depend upon numerous factors.

In the third category (FIG. 5), before transfection with an expressionvector of the present invention, the cell type or cell line expresses aparticular gene product (protein P_(E)) from an endogenous gene, and thecell exhibits transitive RNAi. Following transfection with an expressionvector of the present invention, the transfected cells of the cell typeor cell line express the gene product from both the endogenous gene(protein P_(E)), and from the chimeric RNA transcripts of the presentinvention (protein P_(R)*). Following introduction of the UiRNA, whichtargets the UtRNA of the chimeric RNA transcript, expression of the geneproduct from the chimeric RNA transcripts of the present invention(protein P_(R)*) is silenced, and secondary siRNAs generated during theprimary RNAi response server to silence expression of the gene productfrom the endogenous gene. In this category, following the introductionof the UiRNA, the levels of expression of the gene product under studywill generally drop significantly below the maximum levels of expressionseen following transfection of the transgene, and likely will drop belowthe initial levels observed before the transfection of the transgene.How much below initial levels gene product expression will drop dependsupon how effectively the silencing signal “transits” along the chimericRNA transcript and to corresponding endogenous transcripts to induce asecondary RNAi response. Importantly, if the silencing signal transitsefficiently to endogenous transcripts, and if the gene product understudy is a member of a gene family, the secondary RNAi response may alsolead to the silencing of expression of paralogous genes.

In the fourth category (not depicted), before transfection with anexpression vector of the present invention, the cell type or cell linedoes not express a particular gene product (protein P_(E)) from anendogenous gene, but the cell does exhibit transitive RNAi. Followingtransfection with an expression vector of the present invention, thetransfected cells of the cell type or cell line express the particulargene product from the chimeric RNA transcripts of the present invention(protein P_(R)*). Following introduction of the UiRNA, which targets theUtRNA of the chimeric RNA transcript, expression of the gene productfrom the chimeric RNA transcripts of the present invention (proteinP_(R)*) is silenced. In this category, following the introduction of theUiRNA, the levels of expression of the gene product under study willgenerally drop significantly below the maximum levels of expressiondirected by the transgene, but the amount by which the expression levelis reduced will depend upon numerous factors. As above, if the silencingsignal can transit efficiently to endogenous transcripts, and if thegene product under study is a member of a gene family—other members ofwhich are expressed in this cell type or line—then a secondary RNAiresponse may lead to the silencing of expression of paralogous genes.

Cells that naturally exhibit transitive RNAi include, but are notlimited to the cells of C. elegans and plants. Other cells may beinduced or enabled to exhibit transitive RNAi by, e.g., introducing intothe cells exogenous genes encoding the machinery required for transitiveRNAi. The expected results of methods of the present invention asdescribed above for cells naturally exhibiting transitive RNAi are alsoexpected to be obtained in cells that are made to exhibit transitive.The results expected from practicing the methods in such cell types(i.e., cells that do not normally exhibit transitive RNAi, but are madeto do so) are identical to those described above for the third andfourth categories.

In another set of embodiments, the methods of the present inventionprovide kits for targeting a plurality of gene products for RNAi using asingle UiRNA targeted to a common UtRNA. The methods of the presentinvention further provide kits for determining the effects of alteredlevels of expression of a plurality of gene products, or for assessingthe involvement of a particular gene product, amongst a plurality ofgene products, in pathogenicity, genetic disorders, and infectiousdiseases.

Typically, a kit should contain, in a carrier or compartmentalizedcontainer, reagents useful in any of the above-described embodiments ofthe invention. The carrier can be a container or support, in the formof, e.g., bag, box, tube, rack, and is optionally compartmentalized. Thecarrier may define an enclosed confinement for safety purposes duringshipment and storage. Preferably, instructions for using a kit, and/orreagents contained therein, are also included in the kit.

In one set of kit embodiments, the kits of the present inventioncomprise one or more expression vectors that each comprise an expressioncassette having a multiple cloning site and associated UtRNA, and an RNAthat effectively directs RNAi of the recombinantly expressed geneproducts by targeting the UtRNA in chimeric RNA transcripts expressedfrom these expression cassettes. Using such kits, the nucleotidesequence encoding a particular gene product is conveniently clonedwithin the multiple cloning site of the expression vector, such that theresulting assembled vector directs the expression of a chimeric RNAtranscript that comprises a nucleotide sequence encoding a particulargene product (the subject RNA) and the UtRNA. The RNA that is providedin the kits to direct the RNAi of the recombinantly expressed geneproducts by targeting the UtRNA, can be of any configuration thateffectively directs RNAi by targeting the UtRNA, however, in a preferredembodiment the RNA is a single-stranded shRNA. In an alternativepreferred embodiment the RNA is a double-stranded siRNA.

In another set of kit embodiments, the kits of the present inventioncomprise one or more expression vectors, as described above, plus atranscription vector that directs the in vivo transcription of an RNAthat effectively directs the RNAi of expression of gene products bytargeting the UtRNA borne by the chimeric RNA transcripts encoding thegene product whose expression is to be reduced. In a preferredembodiment this RNA is a single-stranded shRNA. In an alternativepreferred embodiment, this RNA is a double-stranded siRNA.

Further provided by the methods of the present invention are geneproduct panel test kits designed to determine the effects of alteredlevels of expression of a plurality of gene products. In such kits aplurality of expression vectors, such as those described above, areprovided, but these vectors are provided each with a differentnucleotide sequence encoding a specific gene product inserted into it,such that the vector directs the transcription of a chimeric RNAtranscript encoding a specific gene product (the subject RNA) andbearing a common UtRNA. Also provided in these gene product panel testkits would be either RNA that targets the UtRNA and induces RNAi, or atranscription vector that directs the expression of such an RNA.Preferably the RNA is either a small single-stranded hairpin RNA, or adouble-stranded siRNA. Advantageously, these gene product panel kitsinclude at least two expression vectors that direct the expression ofdifferent gene products, but the expression of all gene products encodedin the supplied vectors can be reduced by introducing an RNA designed topromote RNAi by targeting the UtRNA in the chimeric RNA transcriptsencoded in the supplied vectors (i.e., a UiRNA). Preferably these geneproduct panel test kits contain at least 2, 3, 4, 6, 8, 12, 16, 24, 32,36, 40, 48, 56, 60, 64, 72, 80, 84, 88, 96 or 160, 240, 360, 400, 480,560, 600, 1000 or more such expression vectors, each capable ofdirecting the expression of a different gene product, and directing theproduction of a chimeric RNA transcript comprising a UtRNA and thenucleotide sequence encoding a specific gene product (the subject RNA).

Beneficially, the expression vectors of these gene product panel testkits can encode different human gene products that are related insequence or function (i.e., drug metabolizing enzymes, such as mixedfunction oxidases, cytochrome P450 enzymes, and the like), or areinvolved in a single metabolic pathway, or represent a collection ofinteracting proteins that are associated with a particular geneticdisorder or infectious disease.

Alternatively, the expression vectors of these gene product panel testkits may encode different gene products from a human pathogen (e.g.,viral genes from a human viral pathogen, or virulence factor genes froma human parasite), or the expression vectors may encode gene productsfrom a human pathogen that are required for infection of, or maintenancewithin, a non-human species that serves as a vector or reservoir for thedisease (e.g., transmission factor genes of arboviruses or parasiticprotozoa such as Plasmodium falciparum). Also, the expression vectors ofthese gene product panel test kits may encode different gene productsfrom non-human species that serve as vectors for transmission ofinfectious diseases (e.g., genes known or suspected to be required forinfection of Aedes aegypti mosquitoes by West Nile virus). Further, theexpression vectors included in a gene product panel test kit may bedesigned for producing the chimeric RNA transcripts of the presentinvention in human cells, or in the cells of non-human species thatserve as vectors for transmission of infectious diseases.

Alternatively, the expression vectors included in a gene product paneltest kit may be designed for producing the chimeric RNA transcripts ofthe present invention in plant cells. Innumerable such gene productpanel test kits utilizing the methods of the present invention can beenvisioned by skilled artisans appraised of the present invention, andthe examples provided above are not meant to limit the design orapplication of such kits.

In yet another set of embodiments, the methods of the present inventionprovide for arrays designed to utilize RNAi to either simultaneouslymanipulate levels of expression of a plurality of gene products, orsimultaneously determine the effects of altered levels of expression ofa plurality of gene products all by using a single siRNA. For theseembodiments, the methods of the present invention provide for atransfection array of DNA expression vectors, where the expressionvectors at different addresses within the array are capable of directingthe production of different chimeric RNA transcripts that have differentsubject RNAs to be degraded, but bear a common UtRNA. The methods of thepresent invention further provide for arrays of transfected cells,wherein collections of cells at specific addresses within the array aretransfected with a particular DNA expression vector, and this vectordirects the production of a chimeric RNA transcript that encodes aparticular gene product but bears a common UtRNA. In this manner, arraysof cells showing altered levels of expression of a plurality ofparticular gene products are formed, but a single siRNA can be used toinduce RNAi and thereby diminish expression of all gene products encodedby the chimeric RNA transcripts, by introducing into the cells a UiRNA,which corresponds to, and is capable of targeting, the UtRNA common toall of the chimeric RNA transcripts expressed within the cells of thearray.

Such arrays can be microarrays of transfected cells or macroarrays ofcultures of transfected cells. Such arrays can also be macroarrays oforganisms expressing the chimeric RNA transcripts of the presentinvention. Examples of the organisms include nematodes and plants.Nematode and plant cells, tissues, or plant seeds can also be used inthe arrays. For the microarrays, the reverse transfection methodsdescribed by Sabatini in U.S. Pat. No. 6,544,790, which is incorporatedby reference herein in its entirety, can be used to prepare either thetransfection microarrays of expression vectors, or the microarrays ofreverse transfected cells) of these embodiments.

Kits including one or more of the arrays of the present invention and aUiRNA are also contemplated.

Using these embodiments of the present invention (i.e., microarrays ofreverse transfected cells, or macroarrays of transfected cells ororganisms) the levels of expression of a plurality of gene products maybe simultaneously manipulated as required. Initially, and in the absenceof UiRNA, all gene products encoded in the chimeric transcripts of thepresent invention should be overexpressed to some degree. The degrees towhich the gene products are overexpressed can be influenced by any meansknow in the art. For example, different constitutive promoters,exhibiting particular levels of transcriptional activity, can beincorporated into the expression cassettes of the present invention.Promoters exhibiting high transcriptional activity should lead to higherlevels of initial overexpression. Alternatively, inducible orderepressible promoters can be incorporated into the expressioncassettes of the present invention. Such promoters, which are well knownin the art, can be manipulated by addition of particular inducing orrepressing agents to the cell culture medium in order to intentionallyalter their levels of transcriptional activity. Once appropriate levelsof expression of the plurality of gene products under study areachieved, the levels of expression of the plurality of gene products canbe conveniently and simultaneously diminished by the introduction of asingle type of siRNA (UiRNA). Introduction of UiRNA corresponding insequence to the UtRNA found in all chimeric RNA transcripts encoding thegene products under study, induces RNAi, and it's associated silencing(reduction) of expression of all gene products being expressed from thechimeric RNA transcripts of the present invention. The introduction ofUiRNA can be by any means, but the means chosen, as well as the amountintroduced, and the nature of the UiRNA and corresponding UtRNA, willdetermine the amount and duration of silencing (reduced expression)observed.

Whatever the means of introduction of UiRNA, the methods of the presentinvention provide a means to “silence,” or generally diminishexpression, all of the gene products expressed by the cells of thearray. This allows the cells clusters at specific addresses in the arrayto be compared under conditions of increased expression of a particulargene product (in the absence of a UiRNA), and decreased expression of aparticular gene product (in the presence of UiRNA). Since the expressionlevels of the plurality of gene products expressed within the cells ofthe array can be manipulated simultaneously and conveniently by auniform treatment (i.e., the introduction of UiRNA), the methods of thepresent invention are particularly useful for the high-throughputanalysis of gene function, in which large sets of DNAs are screened toidentify those DNAs encoding gene products that cause cellularphenotypes of interest, or exhibit other properties of interest.

Importantly, as described above, cell types or cell lines vary withrespect to their basal levels of expression of a particular gene productor family of gene products. Clearly, the type or line of cells chosenfor practicing the methods of the present invention will have a profoundeffect on the results attained, since different cell types or cell linesexhibiting different basal levels of endogenous expression of variousgene products can be employed to make the microarrays and macroarraysjust described. Generally speaking, and for the purpose of practicingthe methods of the present invention, all cell types can be grouped intoone of four categories, based on whether the cells of that cell type orline express a particular gene product or not, and whether they exhibittransitive RNAi or not. As described above, the methods of the presentinvention can be expected to have different net effects with respect tothe manipulation of expression of a particular gene product dependingupon in which of these four categories the cell type or cell line falls.

In another set of embodiments, the methods of the present inventionprovide a means to reduce the expression of endogenous genes inorganisms or cells that either naturally exhibit transitive RNAi, or aremade to do so. In these embodiments, the coding region of the endogenousgene whose expression is desired to be reduced is cloned within anexpression cassette that also includes a UtRNA and the expressioncassette is included in an expression vector. The coding region isinserted into the expression cassette so that the cassette directs theexpression of a chimeric RNA transcript, comprising the nucleotidesequence encoding the gene product of interest (the subject RNA) and aUtRNA, adjacent to the coding region. For example, if designed to encodea peptide, the UtRNA can be cloned in-frame with the 3′ end of thecoding region, so that the UtRNA directs the addition of a peptide tagto the carboxyl-terminus of the gene product. Alternatively, the UtRNAmay be cloned just downstream of the stop codon, in the 3′ UTR. Theresulting expression vector is then transfected into cells exhibitingtransitive RNAi. Once transfected, these cells express the chimeric RNAtranscript bearing the UtRNA. When reduction of endogenous expression isdesired, a UiRNA corresponding in sequence to the UtRNA is introducedinto the cell. This UiRNA serves as a primary siRNA that induces aprimary RNAi response by targeting the UtRNA in the chimeric RNAtranscript. During the primary RNAi response, secondary siRNAs aregenerated corresponding to sequences 5′ of the UtRNA and in the regionencoding the endogenous gene product. These secondary siRNAs promoteRNAi-induced silencing of the sequences to which they correspond; namelythe coding region 5′ to the UtRNA. Because the sequences that thesesecondary siRNAs target are common to transcripts from bothrecombinantly-expressed and endogenously-expressed genes, a secondaryRNAi response induced by these secondary siRNAs leads to reducedexpression of gene products encoded by both recombinant and endogenoustranscripts. Hence, the methods of the present invention can be used inany cell or organism exhibiting transitive RNAi, whether naturallyexhibited or otherwise induced, to reduce the expression of any geneproduct from endogenous transcripts.

Advantageously, the methods of the present invention can also be used toreduce the expression of highly similar or homologous gene products. Itis known from experiments conducted in organisms and cells exhibitingtransitive RNAi, that siRNAs targeted to a particular member of a genefamily can ultimately induce silencing of other members of that samegene family. The mechanism by which this transitive secondary silencingoccurs is thought to involve the generation of secondary siRNAscorresponding to nucleotide sequences outside of the region ofnucleotide sequences in transcripts originally targeted by the primary(i.e., introduced) siRNAs. (In C. elegans, these secondary siRNAscorrespond to nucleotide sequences 5′ of the nucleotide sequencetargeted by the primary siRNA, but in plants the secondary siRNAscorrespond to nucleotide sequences both 5′ and 3′ of the originallynucleotide targeted sequence.) If these secondary siRNAs correspond tosequences that are shared by gene family members, these secondary siRNAswill also target these gene family members for silencing during asecondary RNAi response. However, in order for gene family members to betargeted by secondary siRNAs, they must possess regions of sequence thatare identical to, or very similar to, regions of sequence adjacent tothe sequence targeted by the original primary siRNAs.

The advantages to using the methods of the present invention, in themanner described above, to reduce expression of endogenous genes incells and organisms exhibiting transitive RNAi, are at least two-fold.First, if a particular UtRNA is incorporated into a plurality ofchimeric RNA transcripts, a single type of siRNA that targets that thatUtRNA (a UiRNA) can be used to reduce the expression of all geneproducts encoded in that plurality of chimeric RNA transcripts. Second,the UtRNA targeted by the UiRNA, and the UiRNA employed, can be selectedand tested in advance, in order to assure that UiRNA-mediated genesilencing works effectively and without regard to the nature of the geneproduct coding sequence included within the chimeric RNA transcripts.

Importantly, however, in order for these embodiments to take advantageof the phenomenon of transitive RNAi to affect silencing of homologousmessages, the UtRNA must be situated adjacent to, and preferably nearby,the nucleotide sequence encoding the gene product whose expression is tobe reduced or silenced. Advantageously, the UtRNA can either be insertedinto the 3′ untranslated region just 3′ of the stop codon, or, if madeto encode a peptide, can be cloned in frame with the 3′ end of thecoding region for the gene product such that the chimeric RNA transcriptproduced from the expression cassette encodes a fusion protein. In thelatter case, it is preferable for the peptide encoded by the UtRNA to bea peptide, which can be used for the ready detection and quantitation ofthe expressed fusion protein.

In still another set of embodiments, the methods of the presentinvention provide a method of treating disease in organisms or cellsthat naturally exhibit transitive RNAi (i.e., nematodes and plants), orin organisms or cells that are made to perform transitive RNAi. In theseembodiments, an expression cassette directing the expression of achimeric RNA transcript, comprising a nucleotide sequence encoding adisease-related protein (as the subject RNA) and a UtRNA, is introducedinto the cells, or organism in need of treatment. The expressioncassette introduced directs the production of a chimeric RNA transcript,which bears the subject RNA and the UtRNA. At some point in time when areduction in the level of expression of the disease-related protein isdesired, a UiRNA, which corresponds in sequence to the UtRNA, isintroduced into the cells or organism to promote a reduction ofexpression of the recombinantly expressed gene-product through a primaryRNAi response. During the course of the primary RNAi response secondarysiRNAs are produced that will correspond to sequences adjacent to, orjust upstream (5′) of the UtRNA. These secondary siRNAs induce asecondary RNAi response that results in the reduction of expression orsilencing of both recombinantly expressed and endogenously expressedgene product. The net reduction of expression of both recombinantlyexpressed and endogenously expressed gene product, desirably promotes animprovement in the disease state.

In a final set of embodiments, the methods of the present inventionprovide a method of treating an infectious disease caused by apathogenic organism that naturally exhibits transitive RNAi. In theseembodiments, transitive RNAi is used to silence, or significantlyreduce, expression of a critical gene product (e.g., a virulence factor)in the pathogenic organism. Examples of pathogenic organisms exhibitingtransitive RNAi include nematode worms, which result in a variety ofdiseases in agricultural crops and livestock, and possibly protozoa,such as trypanosomes, which cause a variety of diseases (i.e., sleepingsickness) in humans and domesticated animals.

3. Expression Cassettes

It will be apparent to skilled artisans that any molecular geneticengineering methods or recombinant DNA technologies may be used in thepresent invention for purposes of preparing the expression cassettes andexpression vectors of the present invention. Generally, once prepared, anucleic acid encoding an expression cassette can be incorporated into anexpression vector or a delivery vector, which is introduced to asuitable target cell.

The expression cassettes employed in the various embodiments describedabove can be of any suitable construction, and can be included in anyappropriate delivery vector. Such delivery vectors include plasmid DNA,viral DNA, and the like. The means by which the expression cassette inits delivery or expression vector is introduced into target cells ortarget organism can be transfection, reverse transfection, virus inducedtransfection, electroporation, direct introduction by biolystics (e.g.,using a “gene gun;” BioRad, Inc., Emeryville, Calif.), and the like.Other methods that can be employed include methods widely known in theart as the methods of gene therapy. Once delivered into a target cell,or target organism the expression cassette may be maintained on anautonomously replicating piece of DNA (e.g., an expression vector), ormay be integrated into the genome of the target cell or target organism.

Typically, to assemble the expression cassettes and vectors of thepresent invention a nucleic acid, preferably a DNA, encoding a geneproduct under study is incorporated into a unique restrictionendonuclease cleavage site, or a multiple cloning site, within apre-existing “empty” expression cassette bearing a UtRNA-encodingsequence to form a complete recombinant expression cassette that iscapable of directing the production of the chimeric RNA transcripts ofthe present invention. These chimeric RNA transcripts comprise a subjectRNA, generally encoding a specific gene product, and the UtRNA, whichcan be targeted by a corresponding UiRNA, to induced the RNAi-mediateddegradation of the chimeric RNA transcript. Frequently such completerecombinant expression cassettes reside within, or inserted into,expression vectors designed for the expression of such chimeric RNAtranscripts, and recombinant proteins. Many types of vectors can be usedfor the present invention to produce chimeric RNA transcripts bearingUtRNAs. Methods for the construction of an expression vector forpurposes of this invention should be apparent to skilled artisansapprised of the present invention. (See generally, Current Protocols inMolecular Biology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. &Wiley Interscience, Ch. 13, 1988; Glover, DNA Cloning, Vol. II, IRLPress, Wash., D.C., Ch. 3, 1986; Bitter, et al., in Methods inEnzymology 153:516-544 (1987); The Molecular Biology of the YeastSaccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. Iand II, 1982; and Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, 1989.)

4. Expression Vectors

Generally, the expression cassettes inserted or assembled within theexpression vectors have a promoter operably linked to a DNA encoding thesubject RNA that is to be manipulated, plus a UtRNA. The promoter can bea native promoter, i.e., a promoter that is responsible for theexpression of that particular gene product in cells, or it can be anyother suitable promoter. Alternatively, the expression cassette can be achimera, i.e., having a heterologous promoter that is not the nativepromoter responsible for the expression of the gene product whoseexpression is to be manipulated. Such heterologous promoters can even befrom a different species than the target cell or organism.

The expression vector may further include an origin of DNA replicationfor the replication of the vectors in target cells. Preferably, theexpression vectors also include a replication origin for theamplification of the vectors in, e.g., E. coli, and selection marker(s)for selecting and maintaining only those target cells harboring theexpression vectors. Additionally, the expression vectors preferably alsocontain inducible or derepressible promoters, which function to controlthe transcription of the chimeric RNA transcript from the DNA thatencodes it. Other regulatory sequences such as transcriptional enhancersequences and translation regulation sequences (e.g., Shine-Dalgarnosequence) can also be operably included in the expression vectors.Transcription termination sequences, and polyadenylation signalsequences, such as those from bovine growth hormone, SV40, lacZ andAcMNPV polyhedral protein genes, may also be operably linked to the DNAencoding the gene product whose expression is to be manipulated.

As mentioned above, the UtRNA, which is also included in the expressionvector, may be situated in any of the five possible locations, withrespect to the subject RNA whose cellular concentration is to bemanipulated, or with respect to the coding sequence of the gene productwhose expression is to be manipulated, as depicted in FIG. 1. In allcases the subject RNA is operably linked to the UtRNA, such that theresulting chimeric RNA transcript is subject to the RNAi-induceddegradation when targeted by an introduced UiRNA that corresponds innucleotide sequence to the nucleotide sequence of the UtRNA. If theparticular embodiment of the invention to be practiced exploitstransitive RNAi to induce the silencing of endogenous or homologoustranscripts, and the transitive RNAi takes place within a nematode, ornematode cell, the coding sequence is preferably placed 5′ of the UtRNA,in one of two possible configurations. In the first of these twopossible configurations, the UtRNA, which is situated adjacent to thecoding region, is designed to encode a peptide sequence, and is placedin frame with the 3′ end of the coding sequence. In the second, theUtRNA is placed in the 3′ untranslated region, just downstream (or 3′)of the stop codon. If the particular embodiment of the invention to bepracticed exploits transitive RNAi to induce the silencing of endogenousor homologous transcripts, and the transitive RNAi takes place within aplant, or plant cell, the UtRNA can be placed in any location within thechimeric RNA transcript, as long as it is operably linked to the subjectRNA.

In a preferred embodiment the UtRNA encodes a detectable peptide, and isoperably linked to the subject RNA by being placed in frame with the 3′end of the coding sequence, such that the expressed chimeric RNAtranscript ultimately results in the translation of a fusion proteinwith a carboxyl-terminal detectable peptide tag.

5. Detectable Peptide Tags

As mentioned above, the UtRNA can encode an detectable peptide tag thatfacilitates detection of an expressed fusion protein bearing that tag.The tag may be an epitope tag that is useful for immunodetection,quantitation, and possibly the purification of the gene product understudy. Such a UtRNA can be operably linked to the DNA encoding the geneproduct whose expression is to be manipulated such that a fusion proteinbearing the epitope tag is expressed. Examples of useful epitope tagsinclude, but are not limited to, influenza virus hemagglutinin (HA),Simian Virus 5 (V5), polyhistidine (His6), c-myc, FLAG™, and the like.Specific antibodies immunoreactive with these epitope tags, and manyothers, are commercially available. Additionally, proteins expressed asfusions with polyhistidine tags can be easily detected and/or purifiedwith, e.g., Ni²⁺ affinity columns.

If the UtRNA of the present invention is designed to encode an epitopetag and is cloned in frame with the nucleotide sequence encoding thegene product whose level of expression is to be manipulated, theresulting fusion protein can be readily detected by Western blotting.Also, the addition of an epitope tag allows the expression level orconcentration of the resulting fusion protein to be determined in asample, such as a cell lysate, without the need for separation,isolation or purification. For this purpose, it is preferred that anantibody selectively immunoreactive with the epitope tag is used in animmunoassay. For example, immunocytochemical methods can be used. Otherwell known antibody-based techniques can also be used including, e.g.,enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA),immunoradiometric assays (IRMA), fluorescent immunoassays, protein Aimmunoassays, and immunoenzymatic assays (IEMA). See e.g., U.S. Pat.Nos. 4,376,110 and 4,486,530, both of which are incorporated herein byreference. These antibody-based techniques are widely known in the artand are described readily available references, such as UsingAntibodies: A Laboratory Manual (Harlow, Cold Spring Harbor Press(1999)).

Alternatively, the UtRNA can encode any other type of detectablepeptide, including, but not limited to, fluorescent peptides, orpeptides exhibiting enzymatic activity. For example, the UtRNA canencode enhanced green fluorescent protein, or any other variety offluorescent protein. Preferably, such fluorescent peptides allow for theready detection of expression of fusion proteins bearing them, and evenmore preferably, they allow for the quantitation of such fusionproteins. Examples of such fluorescent peptides are widely known in theart, and protocols for their detection and quantitation are availablefrom a variety of sources.

Examples of peptides exhibiting enzymatic activity are also widely knownin the art. Such peptides include, but are not limited to, alkalinephosphatase, horseradish peroxidase, and β-galactosidase, which allowfor the ready detection and quantitation of fusion proteins bearing themusing specific enzymatic assays. Examples of such peptides exhibitingenzymatic activity are widely known in the art, and protocols for theirdetection and quantitation are available from a variety of sources.

6. Additional Features

The expression vectors or the present invention may also containcomponents that direct the recombinantly expressed gene product orfusion protein to the surface of the cell, to a particular intracellularcompartment, or to be secreted, as required. Signal peptides, nuclearlocalization sequences, endoplasmic reticulum retention signals,mitochondrial localization sequences, myristoylation signals,palmitoylation signals, and transmembrane sequences are examples ofoptional components that can determine the destination of expressed geneproducts. When it is desirable to manipulate the expression of two ormore gene products in a single host cell by RNAi, two expressioncassettes directing the expression of two different chimeric RNAtranscripts, each encoding a single gene product, in a distinct subjectRNA that is operably linked to a UtRNA, may be incorporated into asingle vector. Alternatively, the two expression cassettes may residewithin two distinct expression vectors, both of which are introducedinto a single target cell. Advantageously, in either scenario, the sameUtRNA may be included in both expression cassettes so that the sameUiRNA can be used to reduce their expression. Alternatively, a differentUtRNA can be incorporated into each expression cassette so that theexpression of the two different gene products can be manipulatedindependently.

The expression vectors of the present invention can be introduced intothe target cells by any techniques known in the art, e.g., by direct DNAtransformation, microinjection, electroporation, viral infection,lipofection, biolystics, and the like. The expression of the geneproducts to be manipulated may be transient or stable, inducible orderepressible. The expression vectors can be maintained in target cellsin an extrachromosomal state, i.e., as self-replicating plasmids orviruses. Alternatively, the expression vectors, or portions thereof, canbe integrated into chromosomes of the target cells by conventionaltechniques such as site-specific recombination or selection of stablecell lines. In stable cell lines, at least the expression cassetteportion of the expression vector is integrated into a chromosome of thetarget cells.

The vector construct can be designed to be suitable for expression invarious target cells, including but not limited to bacteria, yeastcells, plant cells, nematode cells, insect cells, and mammalian andhuman cells. Methods for preparing expression vectors designed forexpression of gene products in different target cells are well known inthe art.

7. Introduction of UiRNA

A universal interfering RNA, or UiRNA, can be introduced into thetransfected target cells of the present invention either exogenously,i.e., from outside the target cells, or endogenously, from transcriptioncassettes that direct the transcription of UiRNAs within the targetcells. For exogenous introduction, the UiRNAs of the present inventionare synthesized in vitro and introduced by transfection, lipofection,electroporation, or any other appropriate means. Such in vitrosynthesized UiRNAs may be synthesized by chemical means, or by enzymaticmeans from the appropriate precursors. In addition, these in vitrosynthesized UiRNAs may take different forms. They may be double-strandedRNA duplexes consisting of two annealed strands of about 21 nucleotidesthat form about 19 basepairs between them and have 3′ overhangs of twonucleotides (Elbashir et al., EMBO J. 20:6877-6888 (2001) & Chiu andRana, Molec. Cell 10:549-561 (2002)). Alternatively, these in vitrosynthesized UiRNAs may be single-stranded short hairpin RNAs (Sui etal., Proc. Natl. Acad. Sci. U.S.A. 99:5515-5520 (2002); Yu et al., Proc.Natl. Acad. Sci. U.S.A. 99:6047-6052 (2002); and Paul et al., NatureBiotech. 20:505-508 (2002)).

For endogenous introduction, UiRNAs are transcribed within the targetcells of the present invention. Such UiRNAs can be of any appropriateform, but are preferably transcribed as small hairpin RNAs that areprocessed to for active UiRNAs by cellular nucleases. Transcriptioncassettes directing the expression of UiRNAs, like the expressioncassettes directing the expression of chimeric RNA transcripts,described above, can be incorporated into a transcription vector or adelivery vector, which is introduced to a suitable target cell.

The expression cassettes employed for the in vivo transcription of UiRNAcan be of any suitable construction, and can be included in anyappropriate delivery vector. Such delivery vectors include plasmid DNA,viral DNA, and the like. The means by which the UiRNA expressioncassette in its delivery or expression vector is introduced into targetcells or target organism can be transfection, reverse transfection,virus induced transfection, electroporation, direct introduction bybiolystics (e.g., using a “gene gun;” BioRad, Inc., Emeryville, Calif.),and the like. Other methods that can be employed include methods widelyknown in the art as the methods of gene therapy. Once delivered into atarget cell, or target organism the UiRNA expression cassette may bemaintained on an autonomously replicating piece of DNA (e.g., anexpression vector), or may be integrated into the genome of the targetcell or target organism.

Typically, to assemble the UiRNA expression cassettes and vectors of thepresent invention a nucleic acid encoding the UiRNA, or a UiRNAprecursor such as an shRNA, is operably linked to a promoter sequencethat directs the transcription of the encoded interfering RNA, orinterfering RNA precursor, by an RNA polymerase. Preferably, thepromoter used in the UiRNA expression cassette is inducible orderepressible and tightly regulated, such that the expression of theUiRNA can be readily controlled. RNA transcription cassettes and vectorssuitable for use in the methods of the present invention are known inthe art and have been described in a variety of references. For example,methods for the in vivo expression of shRNAs are presented in U.S.Patent Application Nos. 2003/0068821, 2003/0139363 and 2003/0144232,which are incorporated herein by reference, in their entirety. Methodsfor the stable expression of interfering RNAs have also been describedin papers by Brummelkamp, et al., (Science 296:550-553 (2002)),Paddison, et al., (Genes and Dev. 16:948-958 (2002)), Sui et al., (Proc.Natl. Acad. Sci. U.S.A. 99:5515-5520 (2002)), and Xia, et al., (NucleicAcids Res. 31: e100 (2003)), which are all incorporated herein byreference in their entirety. Additionally, methods for the establishmentof conditional transcription vectors expressing interfering RNAs inmammalian cells were recently described in a paper by Matsukura et al.,(Nucleic Acids Res. 31:77 (2003)), which is also incorporated herein byreference in its entirety. Finally, a chemical-regulated inducibleinterfering RNA expression system that functions in plants has recentlybeen described in a paper by Guo, et al., (Plant J. 34:383-392 (2003)),which is also incorporated herein by reference in its entirety.

General methods for the construction of such interfering RNAtranscription cassettes and vectors should be apparent to skilledartisans apprised of the present invention. (See generally, CurrentProtocols in Molecular Biology, Vol. 2, Ed. Ausubel, et al., GreenePublish. Assoc. & Wiley Interscience, Ch. 13, 1988; Glover, DNA Cloning,Vol. II, IRL Press, Wash., D.C., Ch. 3, 1986; Bitter, et al., in Methodsin Enzymology 153:516-544 (1987); The Molecular Biology of the YeastSaccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. Iand II, 1982; and Sambrook et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, 1989.)

8. Gene Therapy Techniques

Generally, gene therapy techniques can be used to introduce either theexpression cassettes or vectors of the present invention containing DNAsequences that direct the production of UiRNAs, or their precursors, orboth. Additionally, gene therapy techniques can be used to introduceDNAs that direct the transcription of RNAs (i.e., transcriptioncassettes) that either function as UiRNAs, or serve as precursors (i.e.,shRNAs) that are processed into UiRNAs by cellular enzymes. In one setof embodiments, the levels of expression of a plurality of gene productsis manipulated in cells, tissue, organisms or patients using UiRNAstargeted to the UtRNAs residing in chimeric RNA transcripts transcribedfrom expression cassettes introduced by a gene therapy approach. Forexample, nucleic acids that direct the production of a plurality ofchimeric RNA transcripts encoding a plurality of gene products, orportions or fragments thereof, and bearing a UtRNA, are introduced intocells, tissues, organisms or patients such that the chimeric RNAtranscripts are expressed from expression cassettes provided by theintroduced nucleic acids. Subsequently, or simultaneously, UiRNAs areintroduced into the same cells, tissues, organisms or patients by any ofthe means described above. These UiRNAs target the UtRNAs of thechimeric RNA transcripts and reduce the expression of the encoded geneproduct by directing the degradation of these transcripts. For thesepurposes, nucleic acids encoding a particular gene product, or portionsor fragments thereof, and bearing a UtRNA, especially a UtRNA located 3′of the coding sequence, can be used in the gene therapy approaches inaccordance with the present invention. These embodiments are inherentlyuseful if manipulation of levels of expression of gene products encodedby, and expressed from, transgenes is required. Additionally, in cells,tissues, organisms or patients that naturally exhibit transitive RNAi,or are made to exhibit transitive RNAi, the methods of the presentinvention can also be used to reduce expression from correspondingendogenous genes, and/or homologous genes.

In cells, tissue, organisms and patients not exhibiting transitive RNAi,the methods of the present invention can be used for gene therapy asfollows: if a disease-causing mutation exists an organism or patient orin an endogenous protein gene in cells or tissue in vitro, and ifexpression of a non-mutant form of that gene product is desirable, thena nucleic acid bearing an expression cassette that directs theproduction of a chimeric RNA transcript, including the coding region forthe non-mutant protein and a UtRNA, can be introduced into the cells,tissues, organism or patient. The expression cassette can then be usedto produce a chimeric RNA transcript within the cells, tissues orpatient, and the levels of expression of the non-mutant gene productfrom chimeric RNA transcripts can be further manipulated, by theintroduction of UiRNA that is directed to the UtRNA of the chimeric RNAtranscripts. Advantageously, the expression cassette of the presentinvention can be integrated into the genome, and perhaps can be used toreplace a corresponding defective endogenous gene by, e.g., homologousrecombination. See U.S. Pat. No. 6,010,908, which is incorporated hereinby reference. Alternatively, if the disease-causing mutation is arecessive mutation, the expression cassette is simply used to express awild-type protein from a chimeric RNA transcript, and levels ofexpression of the wild-type protein can be manipulated as required byRNAi induced by introduced UiRNAs that target a UtRNA in the chimericRNA transcript. In another embodiment, if the endogenous genes arenon-mutant but the level of expression of the protein encoded thereby isdesired to be increased, the expression cassette of the presentinvention, encoding the same gene product and bearing a UtRNA can beintroduced into the patient by gene therapy. Once the expressioncassette of the present invention is introduced, the level of expressionof the desired gene product can be further manipulated (i.e.,“fine-tuned”) by the introduction of UiRNAs targeting the UtRNA borne bythe chimeric RNA transcripts expressed from the expression cassettes.

Various gene therapy methods are well known in the art. Successes ingene therapy have been reported recently. See e.g., Kay et al., NatureGenet., 24:257-61 (2000); Cavazzana-Calvo et al., Science, 288:669(2000); and Blaese et al., Science, 270: 475 (1995); Kantoff, et al., J.Exp. Med. 166:219 (1987).

Any suitable gene therapy methods may be used for the purposes of thepresent invention to direct the in vivo production of chimeric RNAtranscripts bearing UtRNAs and encoding desired gene products.Generally, a nucleic acid encoding a desirable gene product and afunctionally linked UtRNA are incorporated into a suitable expressionvector and are operably linked to a promoter in the vector. Suitablepromoters include but are not limited to viral transcription promotersderived from adenovirus, simian virus 40 (SV40) (e.g., the early andlate promoters of SV40), Rous sarcoma virus (RSV), and cytomegalovirus(CMV) (e.g., CMV immediate-early promoter), human immunodeficiency virus(HIV) (e.g., long terminal repeat (LTR)), vaccinia virus (e.g., 7.5Kpromoter), and herpes simplex virus (HSV) (e.g., thymidine kinasepromoter). Where tissue-specific expression of the chimeric RNAtranscript and encoded gene product is desirable, tissue-specificpromoters may be operably linked to the exogenous gene. In addition,selection markers may also be included in the vector for purposes ofselecting, in vitro, those cells that contain the exogenous gene.Various selection markers known in the art may be used including, butnot limited to, e.g., genes conferring resistance to neomycin,hygromycin, zeocin, and the like.

In one embodiment, an transcription cassette directing the transcriptionof the UiRNA, or a precursor thereof, is incorporated into a plasmid DNAvector, which is then introduced into target cells, tissues, organismsor patients. Many commercially available expression vectors may beuseful for the present invention, including, e.g., pCEP4, pcDNAI, pIND,pSecTag2, pVAX1, pcDNA3.1, and pBI-EGFP, and pDisplay.

Various viral vectors may also be used for the methods of the presentinvention. Typically, in a viral vector, the viral genome is engineeredto eliminate the disease-causing capability of the virus, e.g., theability to replicate in the target cells. The exogenous nucleic acid tobe introduced into cells or tissue in vitro or into a patient may beincorporated into the engineered viral genome, e.g., by inserting itinto a viral gene that is non-essential to viral infectivity. Viralvectors are convenient to use as they can be easily introduced intocells, tissues and patients by way of infection. Once in the host cell,the recombinant viral genome is typically is integrated into the genomeof the host cell. In rare instances, the recombinant viral genome mayreplicate autonomously and remain as an extrachromosomal elements.

A large number of retroviral vectors have been developed for genetherapy. These include vectors derived from oncoretroviruses (e.g.,MLV), lentiviruses (e.g., HIV and SIV) and other retroviruses. Forexample, gene therapy vectors have been developed based on murineleukemia virus (See, Cepko, et al., Cell, 37:1053-1062 (1984), Cone andMulligan, Proc. Natl. Acad. Sci. U.S.A., 81:6349-6353 (1984)), mousemammary tumor virus (See, Salmons et al., Biochem. Biophys. Res.Commun., 159:1191-1198 (1984)), gibbon ape leukemia virus (See, Milleret al., J. Virology, 65:2220-2224 (1991)), HIV, (See Shimada et al., J.Clin. Invest., 88:1043-1047 (1991)), and avian retroviruses (See Cossetet al., J. Virology, 64:1070-1078 (1990)). In addition, variousretroviral vectors are also described in U.S. Pat. Nos. 6,168,916;6,140,111; 6,096,534; 5,985,655; 5,911,983; 4,980,286; and 4,868,116,all of which are incorporated herein by reference.

Adeno-associated virus (AAV) vectors have been successfully tested inclinical trials. See e.g., Kay et al., Nature Genet. 24:257-61 (2000).AAV is a naturally occurring defective virus that requires other virusessuch as adenoviruses or herpes viruses as helper viruses. See Muzyczka,Curr. Top. Microbiol. Immun., 158:97 (1992). A recombinant AAV virususeful as a gene therapy vector is disclosed in U.S. Pat. No. 6,153,436,which is incorporated herein by reference.

Adenoviral vectors can also be useful for purposes of gene therapy inaccordance with the present invention. For example, U.S. Pat. No.6,001,816 discloses an adenoviral vector, which is used to deliver aleptin gene intravenously to a mammal to treat obesity. Otherrecombinant adenoviral vectors may also be used, which include thosedisclosed in U.S. Pat. Nos. 6,171,855; 6,140,087; 6,063,622; 6,033,908;and 5,932,210, and Rosenfeld et al., Science, 252:431-434 (1991); andRosenfeld et al., Cell, 68:143-155 (1992).

Other useful viral vectors include recombinant hepatitis viral vectors(See, e.g., U.S. Pat. No. 5,981,274), and recombinant entomopox vectors(See, e.g., U.S. Pat. Nos. 5,721,352 and 5,753,258).

Examples of viral vectors that have already been used to expressinterfering RNAs within mammalian cells include retroviral vectors,lentiviral vectors, adenoviral vectors and adeno-associated viralvectors. The preparation use of these viral vectors for deliver ofinterfering RNA into mammalian cells were described, respectively, inpapers by Barton and Medzhitov (Proc. Natl. Acac. Sci., U.S.A.99:14943-14945 (2002)), Matta et al. (Cancer Biol. Ther. 2:206-210(2003)), Arts et al., (Genome Res. (E-published Sep. 15, 2003)) andTomar et al. (Oncogene 22:5712-5715 (2003)), all of which areincorporated by reference herein in their entirety.

Other non-traditional vectors may also be used for purposes of thisinvention. For example, International Publication No. WO 94/18834discloses a method of delivering DNA into mammalian cells by conjugatingthe DNA to be delivered with a polyelectrolyte to form a complex. Thecomplex may be microinjected into or taken up by cells.

The expression cassette encoding the chimeric RNA transcript or the DNAsequence capable of directing the transcription of a UiRNA, or aprecursor thereof, or a plasmid DNA vector containing such an expressioncassette, may also be introduced into cells by way of receptor-mediatedendocytosis. See e.g., U.S. Pat. No. 6,090,619; Wu and Wu, J. Biol.Chem., 263:14621 (1988); Curiel et al., Proc. Natl. Acad. Sci. USA,88:8850 (1991). For example, U.S. Pat. No. 6,083,741 disclosesintroducing an exogenous nucleic acid into mammalian cells byassociating the nucleic acid to a polycation moiety (e.g., poly-L-lysinehaving 3-100 lysine residues), which is itself coupled to an integrinreceptor-binding moiety (e.g., a cyclic peptide having the sequenceArg-Gly-Asp).

Alternatively, the expression cassette encoding the chimeric RNAtranscript or the DNA sequence capable of directing the transcription ofa UiRNA, or a precursor thereof, or a plasmid DNA vector containing suchan expression cassette can also be delivered into cells via amphiphiles.See e.g., U.S. Pat. No. 6,071,890. Typically, the exogenous expressioncassette, or a vector containing it, forms a complex with the cationicamphiphile. Mammalian cells contacted with the complex can readily takeit up.

The expression cassette encoding the chimeric RNA transcript or the DNAsequence capable of directing the transcription of a UiRNA, or aprecursor thereof, or a DNA vector containing such an expressioncassette, can be introduced into cells or tissue in vitro or in apatient for purposes of gene therapy by various methods known in theart. For example, the expression cassette alone or in a conjugated orcomplex form described above, or incorporated into viral or DNA vectors,may be administered directly by injection into an appropriate tissue ororgan of a patient. Alternatively, catheters or like devices may be usedto deliver exogenous gene sequences, complexes, or vectors into a targetorgan or tissue. Suitable catheters are disclosed in, e.g., U.S. Pat.Nos. 4,186,745; 5,397,307; 5,547,472; 5,674,192; and 6,129,705, all ofwhich are incorporated herein by reference.

In addition, the expression cassette encoding a chimeric RNA transcriptof the present invention, or a DNA sequence capable of directing theexpression of UiRNA, or a precursor thereof, or vectors containing suchexpression cassettes, can be introduced into isolated cells using anyknown techniques such as calcium phosphate precipitation,microinjection, lipofection, electroporation, biolystics,receptor-mediated endocytosis, and the like. Cells containing theexpression cassette and producing the chimeric RNA transcripts of thepresent invention may be selected and redelivered back to the patientby, e.g., injection or cell transplantation. The appropriate amount ofcells delivered to a patient will vary with patient conditions, anddesired effect, which can be determined by a skilled artisan. See e.g.,U.S. Pat. Nos. 6,054,288; 6,048,524; and 6,048,729. Preferably, thecells used are autologous, i.e., cells obtained from the patient beingtreated.

9. Cell Models

In another aspect of the present invention, cell models are provided inwhich the methods of the present invention are used to manipulate thelevels of expression of particular gene products using UiRNAs directedat UtRNAs located within chimeric RNA transcripts expressed within thecells. The cells can be cultured and divided, and one half treated byintroduction of UiRNA, while the other half is sham treated. In this waythe effects of UiRNA-induced RNAi can be evaluated. Alternatively, cellsexpressing the chimeric RNA transcripts of the present invention can becompared with wild type cells under conditions in which levels ofexpression of particular gene products are being manipulated by theintroduction of UiRNAs to both groups of cells. Such cell models areuseful tools for studying cellular functions and biological processesassociated with particular gene products, as well as for detecting anyoff-target effects caused by administration or introduction of UiRNA.Such cell models are also useful tools for studying disorders anddiseases associated with the overexpression or underexpression of geneproducts, both mutant and wild type, and can be used for testing variousmethods for modulating cellular functions, or for treating the diseasesand disorders associated with aberrations in particular gene products oraberrations in their expression levels. Importantly, the gene productswhose levels of expression are manipulated in such cell models, need notbe gene products from cellular genes, but may include gene products fromparasites and pathogens, including viruses.

Advantageously, human cell models in which the methods of the presentinvention are used to manipulate levels of gene expression are providedin accordance with the present invention. Such cell models may beestablished by isolating, from a patient, wild type cells, or cellshaving an aberrant form of one or more gene products, or cellsexhibiting aberrant levels of expression of one or more gene products.The isolated cells may be cultured in vitro as a primary cell culture.Alternatively, the cells obtained from the primary cell culture ordirectly from the patient may be immortalized to establish a human cellline. Any methods for constructing immortalized human cell lines may beused in this respect. See generally Yeager and Reddel, Curr. Opini.Biotech., 10:465-469 (1999). For example, the human cells may beimmortalized by transfection of plasmids expressing the SV40 earlyregion genes (See e.g., Jha et al., Exp. Cell Res., 245:1-7 (1998)),introduction of the HPV E6 and E7 oncogenes (See e.g., Reznikoff et al.,Genes Dev., 8:2227-2240 (1994)), and infection with Epstein-Barr virus(See e.g., Tahara et al., Oncogene, 15:1911-1920 (1997)). Alternatively,the human cells may be immortalized by recombinantly expressing the genefor the human telomerase catalytic subunit hTERT in the human cells. SeeBodnar et al., Science, 279:349-352 (1998).

Alternatively, cell models may be established by isolating, from ananimal or a fungus, wild type cells, or cells having an aberrant form ofone or more gene products, or cells exhibiting aberrant levels ofexpression of one or more gene products. The isolated cells may be fromany type of animal, including fungi, nematodes, insects, and pathogenicprotozoa. The isolated cells may be cultured in vitro as a primary cellculture. Alternatively, the cells obtained from the primary cell cultureor directly from the animal or fungi may be immortalized to establish acell line. Any methods for constructing immortalized cell lines may beused in this respect.

Similarly, cell models may be established by isolating, from a plant,wild type cells, or cells having an aberrant form of one or more geneproducts, or cells exhibiting aberrant levels of expression of one ormore gene products. The isolated cells may be from agriculturallyimportant plants, such as cereal grain plants, and other crop plants,including both monocots and dicots. The isolated cells may be culturedin vitro as a primary cell culture. Alternatively, the cells obtainedfrom the primary cell culture or directly from the plant may beimmortalized to establish a plant cell line. Any methods forconstructing immortalized plant cell lines may be used in this respect.

In alternative embodiments, cell models are provided by recombinantlymanipulating appropriate starting cells. The starting cells may bebacterial cells, yeast cells, fungi cells, insect cells, plant cells,animal cells, and the like. Advantageously, the cells may be derivedfrom mammals, most preferably humans. The starting cells may be obtaineddirectly from an individual, or a primary cell culture, or preferably animmortal stable cell line. In a preferred embodiment, human embryonicstem cells or pluripotent cell lines derived from human stem cells areused as target cells. Methods for obtaining such cells are disclosed in,e.g., Shamblott, et al., Proc. Natl. Acad. Sci. USA, 95:13726-13731(1998) and Thomson et al., Science, 282:1145-1147 (1998).

In one embodiment, a cell model is provided by recombinantly expressinga gene product from the chimeric RNA transcripts of the presentinvention (that also bear a UtRNA), in cells that do not normallyexpress such a gene product. For example, cells that do not contain aparticular gene product may be engineered to express that particulargene product. Alternatively, cells that express a mutant gene productmay be engineered to express a corresponding non-mutant gene productfrom a chimeric RNA transcript that also bears a UtRNA. Simultaneously,or subsequently, UiRNAs can be introduced into these engineered cells toreduce the level of expression of the recombinantly expressed geneproduct. In a specific embodiment, a particular human gene product isexpressed from chimeric RNA transcripts bearing UtRNAs in non-humancells. The cell model may be prepared by introducing into target cellsnucleic acids containing the expression cassettes of the presentinvention, and expressing the encoded gene products in the target cells.For this purpose, the recombinant expression methods described inSections 3-6 may be used. In addition, the methods for introducingnucleic acids into starting cells disclosed in the context of genetherapy in Section 8 may also be used.

In another embodiment, a cell model recombinantly expressing aparticular gene product from the chimeric RNA transcripts of the presentinvention is provided. The over-expression of a gene product from thechimeric RNA transcripts of the present invention, that also bearUtRNAs, may be achieved by introducing into starting cells exogenousnucleic acids containing the expression cassettes of the presentinvention, and selecting those cells that contain the expressioncassettes, produce the chimeric RNA transcripts, and over-express theencoded gene products. The expression of the gene products from theintroduced exogenous nucleic acids may be transient or, preferablystable, but can be manipulated by the introduction of UiRNAs targeted tothe UtRNAs in the recombinantly expressed chimeric RNA transcripts. Therecombinant expression methods described in Section 3-6, and the methodsfor introducing nucleic acids into starting cells disclosed in thecontext of gene therapy in Section 8 may be used. Any cells may beemployed for establishing the cell model. Preferably, human cellslacking the gene product whose expression levels are to be manipulated,or having a normal concentration of the gene product, are used asstarting cells. The starting cells may be obtained directly from anindividual, or a primary cell culture, or preferably a stable immortalhuman cell line. In a preferred embodiment, human embryonic stem cellsor pluripotent cell lines derived from human stem cells are used asstarting cells. Methods for obtaining such cells are disclosed in, e.g.,Shamblott, et al., Proc. Natl. Acad. Sci. USA, 95:13726-13731 (1998),and Thomson et al., Science, 282:1145-1147 (1998).

9.1. Mammalian Cell Models:

In a preferred set of cell model embodiments, mammalian cells are usedas target cells for expression cassettes of the present invention, andfor studies in which the levels of expression of particular geneproducts are manipulated. The expression cassettes in these target cellsdirect the production of chimeric RNA transcripts bearing a UtRNA andencoding a gene product of interest. The chimeric RNA transcripts, inturn, direct the concomitant expression of the gene product of interest,however, the level of expression of the gene product of interest can bemanipulated by the introduction of UiRNA designed to target the UtRNAborne by the chimeric RNA transcripts. For this purpose, virtually anymammalian cells can be used as starting cells including normal tissuecells, stable cell lines, and transformed tumor cells. Conveniently,mammalian cell lines such as CHO cells, Jurkat T cells, NIH 3T3 cells,HEK-293 cells, CV-1 cells, COS-1 cells, HeLa cells, VERO cells, MDCKcells, WI38 cells, and the like are used. Advantageously, mammaliancells that have been manipulated so as to enable the cells to conducttransitive RNAi are used.

Several mammalian expression vectors suitable for directing theexpression of the chimeric RNA transcripts of the present invention arewell known in the art and many are commercially available. These vectorstypically contain a suitable promoter, a multiple cloning site, atranscription termination signal and a polyadenylation signal. Examplesof suitable promoters for the transcription of the chimeric genes inmammalian cells include viral transcription promoters derived fromadenovirus, simian virus 40 (SV40) (e.g., the early and late promotersof SV40), Rous sarcoma virus (RSV), and cytomegalovirus (CMV) (e.g., CMVimmediate-early promoter), human immunodeficiency virus (HIV) (e.g.,long terminal repeat (LTR)), vaccinia virus (e.g., 7.5K promoter), andherpes simplex virus (HSV) (e.g., thymidine kinase promoter). Induciblepromoters can also be used. Suitable inducible promoters include, forexample, the tetracycline responsive element (TRE) (See Gossen et al.,Proc. Natl. Acad. Sci. USA, 89:5547-5551 (1992)), metallothionein IIApromoter, ecdysone-responsive promoter, and heat shock promoters.Suitable origins of replication for the replication and maintenance ofthe expression vectors in mammalian cells include, e.g., the EpsteinBarr origin of replication in the presence of the Epstein Barr nuclearantigen (see Sugden et al., Mole. Cell. Biol., 5:410-413 (1985)) and theSV40 origin of replication in the presence of the SV40 T antigen (whichis present in COS-1 and COS-7 cells) (see Margolskee et al., Mole. Cell.Biol., 8:2837 (1988)). Suitable selection markers include, but are notlimited to, genes conferring resistance to neomycin, hygromycin, zeocin,and the like. Many commercially available mammalian expression vectorsmay be useful for the present invention, including, e.g., pCEP4, pcDNAI,pIND, pSecTag2, pVAX1, pcDNA3.1, and pBI-EGFP, and pDisplay. The vectorscan be introduced into mammalian cells using any known techniques suchas calcium phosphate precipitation, lipofection, electroporation, andthe like.

Similarly, several mammalian transcription vectors suitable fordirecting the transcription of the UiRNAs, or RNAs that are processedinto UiRNAs (i.e., shRNAs), of the present invention are well known inthe art and many are commercially available. Preferably, these vectorscontain an inducible or derepressible promoter functionally linked tothe UiRNA coding sequence, such that expression of the UiRNA, or UiRNAprecursor, is completely absent in the absence of an inducing orderepressing signal, but is present in the presence of an inducing orderepressing signal. In one embodiment, the transcription vectorscontain nucleotide sequences that allow for integration of the vector,or some portion thereof, into the genome of target cells. In anotherembodiment, the vectors possess nucleotide sequences that result intissue-specific transcription, or in the ability to manipulate theexpression levels of the encoded UiRNAs, or UiRNA precursors. An exampleof an inducible siRNA expression system that facilitates tight controlof specific gene silencing by RNAi in human cells was recently describedin a paper by Chen, et al., (Cancer Res. 63:4801-4804 (2003)), which isincorporated herein by reference in its entirety.

Viral expression vectors, which permit introduction of recombinantexpression cassettes into cells by viral infection, can also be used forthe expression of the chimeric RNA transcripts as described above. Viralexpression vectors generally known in the art include viral vectorsbased on adenovirus, bovine papilloma virus, murine stem cell virus(MSCV), MFG virus, and retrovirus. See Sarver, et al., Mol. Cell. Biol.,1: 486 (1981); Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:3655-3659(1984); Mackett, et al., Proc. Natl. Acad. Sci. USA, 79:7415-7419(1982); Mackett, et al., J. Virol., 49:857-864 (1984); Panicali, et al.,Proc. Natl. Acad. Sci. USA, 79:4927-4931 (1982); Cone & Mulligan, Proc.Natl. Acad. Sci. USA, 81:6349-6353 (1984); Mann et al., Cell, 33:153-159(1993); Pear et al., Proc. Natl. Acad. Sci. USA, 90:8392-8396 (1993);Kitamura et al., Proc. Natl. Acad. Sci. USA, 92:9146-9150 (1995);Kinsella et al., Human Gene Therapy, 7:1405-1413 (1996); Hofmann et al.,Proc. Natl. Acad. Sci. USA, 93:5185-5190 (1996); Choate et al., HumanGene Therapy, 7:2247 (1996); WO 94/19478; Hawley et al., Gene Therapy,1:136 (1994) and Rivere et al., Genetics, 92:6733 (1995), all of whichare incorporated by reference.

Generally, to construct a viral vector, a chimeric gene according to thepresent invention can be operably linked to a suitable promoter. Thepromoter-chimeric gene construct is then inserted into a non-essentialregion of the viral vector, typically a modified viral genome. Thisresults in a viable recombinant virus capable of expressing the fusionprotein encoded by the chimeric gene in infected target cells. Once inthe host cell, the recombinant virus typically is integrated into thegenome of the host cell. However, recombinant bovine papilloma virusestypically replicate and remain as extrachromosomal elements.

9.2 Plant Cell and Tissue Models:

In another set of embodiments, the methods of the present invention areconducted in plant cell and tissue systems. Methods for producingchimeric RNA transcripts and expressing exogenous proteins in plantcells and tissues are well known in the art. Similarly, methods forintroducing siRNAs into plant cells and tissues are well known in theart. These methods include the direct introduction of RNAs synthesizedin vitro, as well as the introduction of DNA transcription cassettes andvectors that direct the in vivo production of RNAs. As described above,the RNAs introduced or expressed can be double-stranded siRNAs, orhairpin RNAs that can be processed into siRNAs in vivo, by cellularenzymes. See generally, Weissbach & Weissbach, Methods for PlantMolecular Biology, Academic Press, NY, 1988; Grierson & Corey, PlantMolecular Biology, 2d Ed., Blackie, London, 1988. An example of antightly-regulated chemical-inducible siRNA expression system thatfacilitates the expression of interfering RNAs in plant cells wasrecently described in a paper by Guo, et al., (Plant J. 34:383-392(2003)), which is incorporated herein by reference in its entirety.

Recombinant virus expression vectors based on, e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV) can be also used.Alternatively, recombinant plasmid expression vectors such as Ti plasmidvectors and Ri plasmid vectors are also useful. The expression cassettesof the present invention directing the production of chimeric RNAtranscripts encoding a gene product of interest (in the target RNA) andbearing a UtRNA can be conveniently assembled in expression vectors andplaced under control of a viral promoter such as the 35S RNA and 19S RNApromoters of CaMV or the coat protein promoter of TMV, or of a plantpromoter, e.g., the promoter of the small subunit of RUBISCO and heatshock promoters (e.g., soybean hsp17.5-E or hsp17.3-B promoters).Numerous other methods exist for introducing the expression cassettesand transcription cassettes of the present inventions into plant cells.The use of these methods for the practice of the instant invention willbe apparent to one of skill in the art of manipulating gene expressionin plant cells.

9.3 Insect Cell Models:

In addition, the methods of the present invention can also be conductedin insect cells, e.g., Spodoptera frugiperda, Aedes spp., and Anophelesspp. cells, using any system known in the art. For example,baculovirus-based systems can be used to introduce the expressioncassettes of the present invention to S. frugiperda cells. Expressionvectors and target cells utilizing this system are well known in the artand are generally available from various commercial vendors. Forexample, nucleotide sequences encoding gene products of interestfunctionally linked to UtRNAs can be conveniently cloned into anon-essential region (e.g., the polyhedrin gene) of an Autographacalifornica nuclear polyhedrosis virus (AcNPV) vector and placed undercontrol of an AcNPV promoter (e.g., the polyhedrin promoter). Thenon-occluded recombinant viruses thus generated can be used to infecttarget cells such as Spodoptera frugiperda cells in which the chimericgenes are expressed. See, for example, U.S. Pat. No. 4,215,051.

10. Cell-Based Assays

The cell models of the present invention containing an expressioncassette of the present invention are useful in screening assays foridentifying compounds useful in treating diseases and disorders. Inaddition, they may also be used in in vitro pre-clinical assays fortesting compounds, such as those identified in the screening assays ofthe present invention.

For example, cells may be treated with compounds to be tested andassayed for the compound's activity. A variety of parameters relevant toparticularly physiological disorders or diseases may be analyzed.

11. Transgenic Animals

In another aspect of the present invention, transgenic non-human animalsare created expressing chimeric RNA transcripts encoding particular geneproducts in their subject RNA, and bearing a UiRNA-targetable UtRNA.Animals of any species may be used to generate the transgenic animalmodels, including but not limited to, mice, rats, hamsters, sheep, pigs,rabbits, guinea pigs, preferably non-human primates such as monkeys,chimpanzees, baboons, and the like. Further, animals used to generatetransgenic animal models may be animals that serve as vectors orreservoirs for infectious diseases that infect humans, or commerciallyimportant animals or plants. These animals may be arthropods, such asmosquitoes, fleas and ticks, or they may be from other taxonomic groupsof invertebrates or vertebrates.

In one embodiment, transgenic animals are made to express chimeric RNAtranscripts containing a UtRNA and a subject RNA encoding a gene productwhose expression levels are to be manipulated. Over-expression of thegene products under study may be directed in a cell type or tissue thatnormally expresses the animal counterparts of such gene product.Consequently, the concentration of the selected gene product will beelevated to higher levels than normal. The level of expression can laterbe reduced, if necessary, by the introduction of UiRNAs. Alternatively,one or more gene products are expressed in tissues or cells that do notnormally express such gene products, and the levels of expression ofthese gene products can be manipulated by the introduction of UiRNA.

To achieve over-expression in transgenic animals, the transgenic animalsare made to contain exogenous nucleic acids in the form of expressioncassettes that ultimately direct the production of the chimeric RNAtranscripts of the present invention. These chimeric RNA transcripts, inturn, are translated to yield the gene product, or gene products, understudy. Preferably, the gene products encoded in the expression cassettesare human gene products. Alternatively, the gene products can bevirulence factors of pathogenic organisms, or transmission factors of apathogenic organism's vector species. Such expression cassettes, whichdirect the expression of the chimeric RNA transcripts of the presentinvention, may include a native or non-native promoter, and preferablyan inducible, or derepressible non-native promoter. If the expression ofthe chimeric RNA transcripts is desired to be limited to a particulartissue, an appropriate tissue-specific promoter may be used.

If a reduction of expression of the gene product under study is desired,UiRNAs targeting the UtRNA can be introduced into, or expressed withinthe transgenic animal bearing the expression constructs of the presentinvention. Methods for the introduction of in vitro synthesized UiRNAs,and for the in vivo transcription of UiRNAs are described above. Othermethods of deliver of UiRNAs exist. The efficient delivery of siRNAs toorgans of postnatal mice has been achieved by high pressure tail veininjection (Lewis et al., Nat. Genet. 32:107-108 (2002)). Lentiviralvectors directing the transcription of interfering RNA andtransgenically supplied siRNA have been used successfully to affect aknockdown of gene expression in mice, as described in publications byTiscornia et al. (Proc. Natl. Acad. Sci. U.S.A. 100:1844-1848 (2003))and Hasuwa et al., (FEBS Lett. 532:227-230 (2002), both of which areincorporated herein by reference in their entirety.

In a specific embodiment, the transgenic animal is a “knockout” animalwherein the endogenous gene encoding the animal orthologue of the geneproduct under study is knocked out. The reduced expression, or thecontrolled expression of the endogenous orthologue, may be achieved byknocking out the endogenous gene encoding the gene product under study,typically by homologous recombination. Alternatively, mutations that cancause reduced expression (e.g., reduced transcription and/or translationefficiency, or decreased mRNA stability) may also be introduced into theendogenous genes by homologous recombination. Genes encoding ribozymesor antisense compounds specific to the mRNAs encoding the geneproduct(s) under study may also be introduced into the transgenicanimal. In addition, genes encoding antibodies or fragments thereofspecific to the endogenous protein may also be introduced into thetransgenic animal. Generally, however, the expression levels of aparticular gene product under study are reduced by the introduction of,or in vivo expression of UiRNAs targeted to the UtRNAs born by thechimeric RNA transcript expressed from the expression cassettes of thepresent invention. In a specific embodiment, the lack of expression ofthe animal orthologues of the gene product under study is complementedby expression of an orthologous gene product encoded by a chimeric RNAtranscript of the present invention.

In an alternate embodiment, transgenic animals are made in which theendogenous genes encoding the animal orthologues of the gene productunder study are replaced with the expression cassettes of the presentinvention that direct the expression of chimeric RNA transcripts bearinga UtRNA, and having nucleotide sequences encoding orthologous human geneproducts.

In yet another embodiment, the transgenic animal of the presentinvention expresses specific mutant forms of the gene product understudy. For this purpose, variants of the gene product under studyexhibiting altered activities or properties, and the nucleic acidvariants encoding such variant proteins, may be obtained by random orsite-specific mutagenesis. The transgenic animal of the presentinvention may be made to express such protein variants by modifying theendogenous genes. Alternatively, the nucleic acid variants may beintroduced exogenously into the transgenic animal genome to express theprotein variants therefrom. In a specific embodiment, the exogenousnucleic acid variants are derived from orthologous human genes and thecorresponding endogenous genes are knocked out.

Any techniques known in the art for making transgenic animals may beused for purposes of the present invention. For example, the transgenicanimals of the present invention may be provided by methods describedin, e.g., Jaenisch, Science, 240:1468-1474 (1988); Capecchi, et al.,Science, 244:1288-1291 (1989); Hasty et al., Nature, 350:243 (1991);Shinkai et al., Cell, 68:855 (1992); Mombaerts et al., Cell, 68:869(1992); Philpott et al., Science, 256:1448 (1992); Snouwaert et al.,Science, 257:1083 (1992); Donehower et al., Nature, 356:215 (1992);Hogan et al., Manipulating the Mouse Embryo; A Laboratory Manual, 2ndedition, Cold Spring Harbor Laboratory Press, 1994; and U.S. Pat. Nos.4,873,191; 5,800,998; 5,891,628, all of which are incorporated herein byreference. As mentioned above, methods for creating transgenic miceexpressing interfering RNAs have been described in publications byTiscornia et al. (Proc. Natl. Acad. Sci. U.S.A. 100:1844-1848 (2003))and Hasuwa et al., (FEBS Lett. 532:227-230 (2002), both of which areincorporated herein by reference in their entirety.

Generally, for the purposes of the present invention, the founder linesmay be established by introducing appropriate exogenous nucleic acidsinto, or modifying an endogenous gene in, germ lines, embryonic stemcells, embryos, or sperm which are then used in producing a transgenicanimal. The gene introduction may be conducted by various methodsincluding those described above. See also, Van der Putten et al., Proc.Natl. Acad. Sci. USA, 82:6148-6152 (1985); Thompson et al., Cell,56:313-321 (1989); Lo, Mol. Cell. Biol., 3:1803-1814 (1983); Gordon,Transgenic Animals, Intl. Rev. Cytol. 115:171-229 (1989); and Lavitranoet al., Cell, 57:717-723 (1989). In a specific embodiment, the exogenousgene is incorporated into an appropriate vector, such as those describedabove, and is transformed into embryonic stem (ES) cells. Thetransformed ES cells are then injected into a blastocyst. The blastocystwith the transformed ES cells is then implanted into a surrogate motheranimal. In this manner, a chimeric founder line animal containing theexogenous nucleic acid (transgene) may be produced.

Preferably, site-specific recombination is employed to integrate theexogenous gene or expression cassette into a specific predetermined sitein the animal genome, or to replace an endogenous gene or a portionthereof with the exogenous sequence. Various site-specific recombinationsystems may be used including those disclosed in Sauer, Curr. Opin.Biotechnol., 5:521-527 (1994); Capecchi, et al., Science, 244:1288-1291(1989); and Gu et al., Science, 265:103-106 (1994). Specifically, theCre/lox site-specific recombination system known in the art may beconveniently used which employs the bacteriophage P1 protein Crerecombinase and its recognition sequence loxP. See Rajewsky et al., J.Clin. Invest., 98:600-603 (1996); Sauer, Methods, 14:381-392 (1998); Guet al., Cell, 73:1155-1164 (1993); Araki et al., Proc. Natl. Acad. Sci.USA, 92:160-164 (1995); Lakso et al., Proc. Natl. Acad. Sci. USA,89:6232-6236 (1992); and Orban et al., Proc. Natl. Acad. Sci. USA,89:6861-6865 (1992).

The transgenic animals of the present invention may be transgenicanimals that carry a transgene in all cells or mosaic transgenic animalscarrying a transgene only in certain cells, e.g., somatic cells. Thetransgenic animals may have a single copy or multiple copies of aparticular transgene.

The founder transgenic animals thus produced may be bred to producevarious offspring. For example, they can be inbred, outbred, andcrossbred to establish homozygous lines, heterozygous lines, andcompound homozygous or heterozygous lines.

12. Transgenic Plants

In another aspect of the present invention, transgenic plants arecreated expressing chimeric RNA transcripts comprising a subject RNA,which encodes a particular gene product, and a UtRNA. Plants of anyspecies, monocot or dicot, may be used to generate the transgenic plantmodels. Such transgenic plant models may include, but are not limitedto, Arabadopsis thaliana, cereal and other grain plants (e.g., maize,rice, barley and wheat), other crop plants including vegetable plants,fruit trees, tuber yielding plants (e.g., potatoes, sweet potatoes, andcassaya), tobacco, ornamental plants, and the like.

Methods for the production of transgenic plants are well known in theart. (See Jones & Sutton, eds. Plant Molecular Biology: EssentialTechniques, John Wiley & Sons, Ltd., London (1997); Martinez-Zapater &Salinas, eds., Arabidopsis Protocols, Methods in Molecular Biology,Volume 82, Humana Press, Totowa, N.J. (1998); and Gilmartin & Bowler,eds. Molecular Plant Biology, Vol. 1, Oxford University Press, London(2002).) For example, the transgenic plants of the present invention canbe created through agrobacterium-mediated transformation, using Ti- orRi-plasmid into which the desired sequence is inserted. For the purposesof the present invention, the expression cassettes of the presentinvention that direct the expression of chimeric RNA transcripts can beinserted into the Ti- or Ri-plasmids, which can then be propagated inagrobacterium, and ultimately introduced into a host plant byagrobacterium-mediated transformation. Alternatively, the expressioncassettes of the present invention can be introduced directly into plantcells by biolystics (i.e., gene gun), or into plant cell sphereoplastsby electroporation.

Regardless of the means of introduction, the introduced expressioncassettes then direct the production of chimeric RNA transcripts bearinga UtRNA and a subject RNA encoding a gene product whose expressionlevels are to be manipulated. These chimeric RNA transcripts can then betranslated to direct the overexpression of the gene product of interest.At some point in time when a reduction of expression of the encoded geneproduct is desired, a UiRNA can be introduced into, or synthesizedwithin, the cells or tissues of the transgenic plant. The UiRNA, whichcorresponds in sequence to the UtRNAs of the chimeric RNA transcripts,induces RNAi (also known as post-transcriptional gene silencing, or PTSGin plants) by directing the degradation of the chimeric RNA transcripts.In species that exhibit transitive RNAi, secondary siRNAs producedduring the initial, or primary RNAi response can target transcripts ofrelated sequence for degradation, including transcripts produced fromthe plant's corresponding endogenous gene(s) (i.e., endogenoustranscripts).

Particularly useful methods for producing transgenic plants that can beused for practicing the methods of the present invention have beendescribed recently by Guo and coworkers (Guo, et al., Plant J.34:383-392 (2003)). In particular, Guo and colleagues have developed achemical-inducible Cre/loxP recombination system to trigger theexpression of an intron-containing inverted-repeat inhibiting RNAplants. They have demonstrated its use in producing transgenicArabidopsis thaliana and Nicotiana benthamiana plants, in which the invivo transcription of inhibiting RNAs is not only induced at will, butis also stringently controlled. The methods presented can be used forthe present invention, by redesigning the expression system to produceUiRNA, and preparing transgenic plants in which UiRNA production isstringently controlled but inducible at will. These transgenic plants,or tissues or cells isolated from them, can then be transfected with theexpression vectors of the present invention that direct the expressionof chimeric RNA transcripts bearing a subject RNA, encoding the geneproduct of interest, and a UtRNA. In such transfected transgenic plants,or plant tissues or cells, induction of UiRNA expression will result insilencing of the gene product encoded by the subject RNA through aprimary RNAi response. During this primary RNAi response, secondarysiRNAs will be produced that can affect a secondary, transitive RNAi ofgenes comprising regions of homologous nucleotide sequence.

13. Arrays

In another aspect of the present invention, the methods disclosed hereinprovide a strategy for the high throughput analysis of gene function intransfected cells, especially in transfected cells that either naturallyexhibit, or are made to exhibit, transitive RNAi. In such arrays,introduction of the UiRNA initiates the RNAi-mediated destruction of thesubject RNA carried on the chimeric RNA transcript expressed from theresident expression vector. Similarly, arrays of transfected ortransgenic tissues or organisms are contemplated, wherein theintroduction of the UiRNA to individual members of the array results inthe RNAi-mediated destruction of a distinct subject RNA. In this manner,a plurality of genes can be subjected to RNA interference at the sametime, through the introduction of a single UiRNA. Additionally, in orderto prepare such arrays of cells, tissues, or organisms, arrays ofexpression vectors, or viral delivery vectors are also contemplated.

Preferably these arrays contain a plurality of addresses characterizedby their association with a distinct subject RNA, whether that subjectRNA is merely encoded within a naked DNA expression vector, or is beingexpressed within a transfected cell, tissue or organism. Preferablythese arrays contain at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 18,24, 48, 96, 200, 500, 1000 or more distinct addresses, eachcharacterized by an association with a distinct subject RNA.

13.1. Microarrays

In one embodiment, methods of the present invention provide fortransfection microarrays. As used herein, the term “transfectionmicroarray” refers to solid surface of at least 4 cm², upon which aplurality of spots, each with a diameter of at least 50 μm, are printedand subsequently dried or congealed in a defined regular grid pattern(“array”), where each spot initially contains at least 0.5 nl of anaqueous mixture of a nucleic acid, or nucleic acids, and a compound(e.g., gelatin) that will create a protective but semipermeable matrixaround the nucleic acids when said matrix is dried or congealed, andwhere the individual spots within the array are separated from eachother by at least 200 μm. (For examples of transfection microarrays andthe methods by which they are prepared, refer to U.S. Pat. No.6,544,790, which is incorporated by reference herein in its entirety.)

The term “transfected cell microarray,” as used herein, refers to smallclusters of at least 10 cells growing on the surface of a previouslyprepared “transfection microarray,” each cluster of cells having beentransfected by the nucleic acid or nucleic acids originally containedwithin the matrix of the spot upon which they grew. Said clusters oftransfected cells may, or may not, reside within a monolayer, orcontiguous “lawn” of cells in which the cells growing betweentransfected clusters are not themselves transfected. (For examples oftransfected cell microarrays and the methods by which they are prepared,refer to U.S. Pat. No. 6,544,790, which is incorporated herein byreference.)

As used herein, the term “reverse transfection” refers to the techniquewhereby nucleic acids, in the form of a transfection microarray treatedto facilitate transfection, are placed in a cell culture container, intowhich living cells are subsequently introduced and allowed to grow as amonolayer on the surface of the transfection microarray, and wherebysmall clusters of cells growing in direct contact with specificmicroarray spots take up and become transfected with the specificnucleic acid, or nucleic acids, contained within that particularmicroarray spot. (For details on how reverse transfections areconducted, refer to U.S. Pat. No. 6,544,790, which is incorporatedherein by reference.)

Using the methods disclosed by Sabatini in U.S. Pat. No. 6,544,790,which is incorporated by reference herein in it entirety, transfectedcell microarrays can be created that are suitable for inducing thesilencing, or reduction of expression, of large sets of gene productsusing a common UiRNA. Using a coated glass slide, or other suitablesolid substrate, that is printed with sets of expression vectors, eachvector containing an expression cassette that directs the expression ofa chimeric RNA transcript comprising an subject RNA encoding aparticular gene product, and bearing a UtRNA, a living microarray ofcell clusters recombinantly expressing the gene products, can begenerated. Once generated, a UiRNA corresponding in sequence to thecommon UtRNA can be introduced to the cells of the transfected cellmicroarray to induce RNAi, to simultaneously reduce the expression ofall gene products encoded by the set of expression vectors.

Any cell type may be used to make such arrays, as long as it can beefficiently transfected with an expression vector, and can tolerate theintroduction of UiRNA to induce RNAi. For the purpose of theseembodiments of the present invention, UiRNAs can be introduced into thecells of the transfected cell microarrays by any effective means. Forexample, UiRNAs synthesized in vitro can be introduced by transfectionusing lipofectamine 2000 (Invitrogen, Carlsbad, Calif.). Such invitro-synthesized UiRNAs may be single-stranded shRNAs, or doublestranded siRNA duplexes. In an alternative embodiment, the cells of thetransfected cell microarrays can also be transfected with transcriptionvectors that direct the in vivo production of UiRNA. Alternatively, thecells can be transgenic cells capable of regulated, inducible synthesisof UiRNA upon treatment with an inducing agent. UiRNAs within such cellsmay take the form of double-stranded siRNA duplexes, or single strandedshRNAs.

In a preferred alternative embodiment, cells used to prepare thetransfected cell microarrays of the present invention are modified inadvance to contain an integrated inducible, or derepressible, expressioncassette, or cassettes, that can be made to direct the synthesis ofUiRNAs when provoked with a specific signal. Such cells may expresseither a single-stranded shRNA that is processed into a UiRNA bycellular nucleases, or two single-stranded RNAs that anneal to form aUiRNA. The advantage of such cells is that they are poised for inductionof RNAi, and minimal manipulation is required for them to beginexpressing the UiRNA that is targeted to the UtRNA of the chimeric RNAtranscripts produced by expression vectors subsequently introduced intothe cells. In particular, the methods described in a paper by Guo andcoworkers (Guo, et al., Plant J. 34:383-392 (2003)), which isincorporated by reference herein in its entirety, can be used for thepractice of the present invention in plant cell microarrays.

If the cells employed to make these transfected cell microarrays exhibittransitive RNAi, secondary siRNAs generated during the primary RNAiresponse (induced by the introduction of UiRNAs), will direct thesilencing of endogenously expressed gene products that correspond insequence to the recombinantly expressed gene products encoded by thechimeric RNA transcripts expressed. As indicated above, cells naturallyexhibiting transitive RNAi can be used to prepare such transfected cellmicroarrays, as well as cells that are made to exhibit transitive RNAi.

In another set of embodiments, the living microarrays of transfectedcell clusters can be used to determine or confirm what gene products areresponsible for particular cellular functions, such as thedetoxification of specific toxicants, or the metabolism of specificdrugs. In these embodiments the set of DNAs inserted into the expressioncassettes of the present invention are specifically chosen because oftheir suspected or known role in the phenomenon under study. Generallyfor such purposes, two identical microarrays of transfected cells areprepared and handled as a matched set. Within each set, one array servesas the treated experimental (test) array, while the other serves as anon-treated control. In such a matched set, specific gene products areoverexpressed at defined locations or addresses within the arrays byclusters of cells. Exposure of the test array to the toxicants or drugsmay occur before, after, or during the recombinant expression of thegene products under study.

In a related set of embodiments, the living microarrays of transfectedcell clusters can be used to identify or confirm what pathogen or hostgene products are required for infection by selected pathogenic agents,or what pathogen gene products contribute most to infectivity orpathogenicity. For example, the viral gene products required for viralinfection, viral replication, or viral egress, can be identified.Alternatively, host cell gene products exploited by a pathogen to invadethe host, or responsible for a host's resistance to infection, can alsobe identified.

Advantageously, the cells comprising living microarrays of transfectedcell clusters used in such studies need not be human cells, mammaliancells or even animal cells. In fact, the cells used can be isolated fromvector species that serve as reservoirs for infectious agents. Forexample, a living microarrays of transfected mosquito cell clusters canbe employed to study what gene products are critical for the invasion orsurvival of a particular pathogen in mosquitoes, and the gene productstested may be host (mosquito) cell gene products, or pathogen (i.e.,Plasmodium faciparum, dengue virus, West Nile virus) gene products.

Additionally, the living microarrays of transfected cell clustersexpressing different gene products can be examined before, after, andduring the introduction of UiRNA, and can be screened for clusters ofcells that exhibit detectable differences from other clusters oftransfected cells in the same array, or from non-transfected controlcells in the same array. Alternatively, two duplicate living microarraysof transfected cell clusters can be prepared, and RNAi can be induced inone microarray by the introduction of UiRNA, while the second microarrayserves as a non-treated, or sham-treated, control.

13.2. Macroarrays

Alternatively, macroarrays of cells transfected with the expressioncassettes of the present invention, or even macroarrays of transgenicorganisms bearing expression cassettes of the present invention, can beprepared. In such macroarrays, the cells or organisms at each separateaddress within the macroarray carry a unique expression cassette of thepresent invention, that expression cassette being capable of directingthe expression of a chimeric RNA transcript of the present invention.Given such macroarrays, the methods of the present invention allow forthe ready manipulation of expression levels for all of the gene productsencoded in the chimeric RNA transcripts, by the introduction of the sameUiRNA to all addresses within the macroarray.

Such macroarrays of transfected cells can be prepared in multi-welledplastic containers, such as standard 6 well culture plates, or 96microwell plates, and any other readily-manipulated configurations. Thetransfected cells in such macroarrays may be grown in suspension, or inmonolayer culture. The size and configuration of such macroarrays willbe optimally chosen depending upon the number of cells required forsubsequent assays, and the number of gene products under investigation.Such macroarrays can comprise 2, 3, 4, 6, 12, 18, 24, 48, 96, 120, 180,240, 480, 960 or more clusters of transfected test cells.

For macroarrays of transgenic organisms, arrays can be produced byarranging containers suitable for the culture of such organisms, in aregular configuration with defined addresses. Such arrays may consist oftest tubes arranged in a rack, or culture vessels (e.g., flower pots)arranged on a tray, and such arrays may comprise 2, 3, 4, 6, 12, 18, 24,48, 96, 120, 180, 240, 480, 960 or more transgenic organisms.

In certain embodiments, a library of expression cassette can be providedin which each expression cassette encodes a chimeric RNA transcript asdescribed above comprising a subject RNA operably linked to a universaltarget RNA. The library should encode at least 2, 5, 6, 7, 8, 9, 10, 15,20, 30, 40, 50, 100, 200, 500, 100, 5000, 10,000, 20,000, 30,000, 50,000different subject RNAs while all chimeric transcripts encoded by thelibrary have a common universal target RNA (UtRNA). The subject RNAs maycorrespond to a collection of genes of interest, e.g., a subset of genesof an organism (e.g., virus, nematode, drosophila, animal or human), orsubstantially all genes of an organism (e.g., virus, nematode,drosophila, animal or human).

The expression cassettes in the library may be in expression vectors(e.g., plasmid vector, viral vector, etc.) or simply in linear strandDNA. In addition, the expression cassettes in the library can bearranged in an addressable array (e.g., on a solid support), and theidentity of the subject RNA encoded by the expression cassette in eachaddressable spot in the array can be either known or unknown.Alternatively, the library is a mixture of different expressioncassettes encoding different chimeric RNA transcripts.

In one embodiment, the arrayed library can be used to produce an arrayof target cells as described elsewhere in the present disclosure.

In another embodiment, the library in a mixture is introduced into aplurality of cells or tissues or organisms to produce a plurality ofmixed target cells for expressing therein the chimeric RNA transcripts.As such, upon inducing RNA interference with a universal interfering RNAtargeting the UtRNA in the chimeric RNA transcripts, the phenotypes ofeach target cell exhibited under certain conditions reflect the effectof the subject RNA expressed in the target cell. Therefore, byidentifying and isolating the cells exhibiting the phenotypes ofinterest, the identity of the subject RNA in the target cell can bedetermined and the function of the corresponding gene can be deciphered.Preferably, the library and the cells, tissues or organisms arecontacted in a ratio such that approximately each cell receives onemolecule of expression cassette. The cells can be physically separated,e.g. by spreading onto a medium support, in a similar manner to themethod of cDNA library screening in bacteria or yeast host cells. Thetarget cells are then subject to RNA interference with the universalinterfering RNA described above, and also subject to certain conditions.The cells exhibiting phenotypes of interest upon RNA interference can beisolated and the expression cassette contained therein is characterizedthereby associating the gene in the expression cassette with thecellular phenotype.

It is noted that in these embodiments, kits are also contemplatedincluding the library (arrayed or disarrayed) or target cells (arrayedor disarrayed), and a universal interfering RNA targeting the universaltarget RNA in the library (or a vector expressing the interfering RNA),and optionally instructions for using the kits in the manner describedabove.

In alternative embodiments, the host cells used in making the targetcells described above contain a transcription vector expressing theuniversal interfering RNA, preferably under certain inducing conditions.As such, the target cells are produced by introducing the expressioncassettes or library of expression cassettes into the host cells. Eachtarget cell produced in this manner contain both the transcriptionvector for expressing the universal interfering RNA and an expressioncassette for expressing a chimeric RNA transcript having a subject RNAoperably linked to a universal target RNA upon which the universalinterfering RNA targets. The nucleic acid in the transcription vectorencoding the universal interfering RNA can be integrated into thechromosome of the target cells. Preferably, the nucleic acid in thetranscription vector or integrated in the chromosome is operably linkedto an inducible promoter such that the expression of the universalinterfering RNA is inducible under certain defined conditions. Anyinducible promoters known in the art may be used.

Thus, the present invention also encompasses a plurality of target cellseach expressing a chimeric RNA transcript that has a subject RNAoperably linked to a universal target RNA, wherein at least 2, 4, 5, 6,7, 8, 9, 10, 12, 15, 20, 32, 48, 96, 200, 500, 1,000, 5,000, 10,000 or30,000 or more of the plurality of target cells have different subjectRNAs, and wherein all of the plurality of target cells have the sameuniversal target RNA. In addition, each of the plurality of the targetcells expresses a universal interfering RNA targeting the universaltarget RNA. Kits comprising such plurality of target cells are alsocontemplated. The kits may further comprise instructions for using thekits in the various methods of the present invention.

14. Disease Applications

The methods of the present invention are useful for investigatingdiseases and disorders characterized by altered levels of expression ofparticular gene products, or involving the expression of particularvirulence or transmission factors of pathogenic organisms. The methodsare particularly useful in the study of diseases that either (a) infectorganisms that exhibit transitive RNAi (e.g., plants), (b) requiretransmission through vector species that exhibit transitive RNAi, or (c)are caused by organisms that exhibit transitive RNAi (e.g., nematodesand possibly pathogenic protozoa). In each of these scenarios, themethods of the present invention can be used to manipulate the levels ofexpression of host gene products, such as those host gene products thatare used by pathogens to recognize or invade target cells (i.e., cellsurface receptors). The methods of the present invention can also beused to manipulate the levels of expression of pathogen gene products,such as gene products that, when expressed by pathogens, cause anincrease in virulence of the pathogen (i.e., virulence factors).Additionally, in scenarios where a pathogen infects a transmissionvector species, or intermediate host, (i.e., Plasmodium spp. orArboviruses in mosquitoes), and the transmission vector or intermediatehost species is responsible for transmission of the pathogen to humans,the methods of the present invention can be used to manipulate levels ofexpression of pathogen gene products required for the infection of thetransmission vector, or transmission of the pathogen from theintermediate host to humans (i.e., “transmission factors”). When eitherthe pathogen or intermediate host exhibits transitive RNAi, the methodsof the present invention can potentially be used to reduce theexpression levels of both recombinantly expressed gene products, as wellas the corresponding, or homologous, endogenously expressed geneproducts, as described above. In such cases the methods of the presentinvention may provide a mechanism to block the infectious cycles ofpathogenic organisms before they infect their ultimate hosts.

Advantageously, the methods of the present invention can be used tosimultaneously investigate the involvement of numerous gene products, ofboth host and pathogen, in the course of infection by infectious diseaseagents. In order to conduct such investigations, microarrays oftransfected cells, or even macroarrays of transgenic organisms, intowhich an expression cassette of the present invention has beenintroduced, can be prepared. The expression cassettes direct theproduction of chimeric RNA transcripts that comprise a subject RNA,encoding a particular gene product under study, and a UtRNA. Whentranslated, the chimeric RNA transcripts direct the overexpression ofthe particular inserted gene product. When expression of the insertedgene product is to be reduced in all members of the array, the sameUiRNA is introduced into, or transcribed within, the cells or organismsof the array. The UiRNA, which corresponds in sequence to the commonUtRNA incorporated into the chimeric RNA transcripts, induces a primaryRNAi response and causes the selective degradation of the chimeric RNAtranscripts in the cells or organisms of the array. In cells ororganisms that naturally exhibit transitive RNAi, or are made to exhibittransitive RNAi, secondary siRNAs produced during the primary RNAiresponse correspond in nucleotide sequence to regions nearby the UtRNAin the originally targeted chimeric RNA transcripts. These secondarysiRNAs can induce a secondary RNAi response, and promote the degradationof the transgenically-expressed chimeric RNA transcripts, as well asendogenous transcripts bearing homologous nucleotide sequences.Consequently, introduction of UiRNAs in such cells or organisms, whethersynthesized in vitro or transcribed in vivo, can lead to greatly reducedexpression of the recombinantly expressed, as well as endogenouslyexpressed, gene product under study. In essence, and with regard to thegene product under study in that particular cluster of cells or organismin the array, the result can be a silencing (functional knockdown orknockout), or at least a partial reduction of expression of a particulargene product. Consequently, the arrays described above can be used tosimultaneously investigate the role of a plurality of specific geneproducts in all aspects of disease etiology, or in all stages in thecycle of an infectious disease including pathogen invasion, replication,egress and transmission to new hosts.

The diseases that can be studied by the methods of the present inventioninclude genetic disorders, metabolic disorders, and infectious diseasescaused by any class of pathogen, including, e.g., viruses, bacteria,fungi and protozoa. These diseases may affect humans or non-humananimals, or may affect plants of any variety.

Also, as described above, the methods of the present invention can beused to study the infection of non-human intermediate hosts ortransmission vector species, such as those arthropods that transmitinfectious diseases to humans (e.g., mosquitoes, ticks, fleas, and thelikes). For these studies, arrays of transfected arthropod (i.e.,mosquito) cells may be employed.

The methods of the present invention can also be used to study the geneproducts involved in the infection of plants by various plant pathogens,including viruses, bacteria, fungi, nematodes, and other plantpathogens. For these studies, microarrays of transfected plant cells maybe employed, or macroarrays of transgenic plants may be used.

Advantageously, since plants exhibit transitive RNAi, the methods of thepresent invention can be used to create and select disease- orpathogen-resistant transgenic varieties, or alternatively, can be usedto develop new means to protect plants from infection by pathogens. In aparticularly preferred embodiment of the present invention, a pluralityof transgenic plants are prepared, in which the subject RNAs of thechimeric RNA transcripts correspond to mRNAs, or fragments of mRNAsobtained from, or expressed by a particular plant pathogen. Uponintroduction of the UiRNA to these transgenic plants, preferably by theinduction of in vivo transcription of the UiRNA, a primary RNAi responsewill be mounted. During this primary RNAi response, secondary siRNA withnucleotide sequences corresponding to regions of the subject RNA will begenerated, within each plant. Since each transgenic plant expresses adifferent chimeric RNA transcript with a distinct subject RNA, adifferent set of secondary siRNAs, will made in each plant. Asconcentration of secondary siRNAs nears its peak, the plurality oftransgenic plants can be exposed to the pathogen that served as theoriginal source of the subject RNAs. Those transgenic plants containingcertain preferred sets of secondary siRNAs should be more resistant tothe pathogen than those transgenic plants containing sets of siRNAswhich are ineffective at blocking infection by the pathogen. Thetransgenic plants that are most resistant to infection by the pathogenare selected and the subject RNA expressed within them is identified.The subject RNA thus identified corresponds to the pathogen mRNA, ormRNA fragment, that should be specifically targeted by gene-specificsiRNAs expressed in subsequently produced disease-resistant, transgenicplants.

Such gene-specific siRNAs thus identified can be expected to beespecially effective against infection, if the pathogen under study is anematode. This is because nematodes have been shown to (1) take upsiRNAs from the food they eat, (2) amplify the silencing signal, and (3)carry out transitive RNAi. Consequently, the methods of the presentinvention are particularly well-suited to identifying siRNAs that can beexpressed within plant tissues that will impart resistance to attack ofthese tissues by nematodes. However, the methods of the presentinvention are clearly not limited to developing nematode-resistantplants. Indeed the subject RNAs of the chimeric RNA transcriptsexpressed within a plurality of transgenic can correspond in sequence toRNAs from any plant pathogen. And since siRNAs have recently been shownto mediate the interference of viral DNA accumulation in Nicotianatabacum (Vanitharani et al., Proc. Natl. Acad. Sci. U.S.A.100:9632-9636), the methods of the present invention will likely proveuseful in identifying siRNAs that, when expressed within transgenicplants, impart resistance to viral infections.

Advantageously, the methods of the present invention also can be used tocreate transgenic plants that are resistant to infection by any numberof pathogens. Specifically, once subject RNAs from several differentpathogens are identified that result in the generation of secondarysiRNAs effective in imparting resistance to that pathogen, transgenicplants expressing several different chimeric RNA transcripts, each witha different preferred subject RNA, but the same UtRNA can be prepared.

Importantly, RNAi has now been documented in a large number of humanpathogens, including members of the family of flagellate protozoaTrypanosomatidae (Robinson & Beverly. Mol. Biochem. Parasitol.128:217-228 (2003); Huynh et al., J. Biol. Chem. (Epub Jul. 29, 2003);Tschudi et al., Methods 30:304-312 (2003)), the parasitic flatwormSchistosoma mansoni (Boyle et al., Mol. Biochem. Parasitol. 128:205-215(2003)), and the human filarial nematode parasite Brugia malayi(Aboobaker AND Blaxter Mol. Biochem. Parasitol. 129:41-51 (2003).Furthermore, stable and heritable RNAi has been achieved in the malariavector mosquito Anopheles stephensi (Brown et al., Nucleic Acids Res.31:e5 (2003)), and intravenous injection of siRNAs designed to silence aPlasmodium berghei (mouse malaria) gene into the bloodstreams of mice,affected the silencing of that gene within circulating malarialparasites (Mohmmed et al., Biochem. Biophys Res. Commun. 309:506-511(2003)). These studies, and studies like these, provide support to thenotion that the methods of the present invention can be useful indeveloping novel approaches to utilize RNAi as a means to combating theinfectious diseases of humans. For example, as described above, themethods of the present invention can be used to simultaneously screenthe effects of silencing a plurality of genes from human pathogens,using a single UiRNA to affect the silencing of all genes. In thisfashion, specific genes from important human pathogens can be identifiedas viable targets for therapeutic gene-specific silencing bygene-specific interfering RNAs.

Finally, a recent report by Vanitharani and coworkers (Proc. Natl. Acad.Sci. U.S.A. 100:9632-9636 (2003)), which is incorporated by reference inits entirety, documents the successful siRNA-mediated suppression ofgene expression in cultured plant cells, and demonstrates that siRNAscan interfere with and suppress the accumulation of a nuclear-replicatedDNA virus. This report, in conjunction with the previously describedchemical-regulated inducible RNAi system developed by Guo and colleagues(Guo, et al., Plant J. 34:383-392 (2003)), provide support to the notionthat the methods of the present invention can be useful in developingnovel approaches to utilize RNAi as a means to combating the infectiousdiseases of plants.

15. Pharmaceutical Compositions and Formulations

In another aspect of the present invention, pharmaceutical compositionsare also provided containing the UiRNAs of the present invention, orRNAs that are processed into UiRNAs by cellular enzymes. Thecompositions are prepared as a pharmaceutical formulation suitable foradministration into a patient or infected organism. Accordingly, thepresent invention also extends to pharmaceutical compositions,medicaments, drugs or other compositions containing one or more of thetherapeutic agent in accordance with the present invention.

For example, such therapeutic agents include, but are not limited to,(1) double-stranded UiRNAs, (2) small single-stranded hairpin RNAs thatare processed into UiRNAs by cellular enzymes, (3) DNA vectors thatdirect the transcription of double-stranded UiRNAs, (4) DNA vectors thatdirect the transcription of small single-stranded hairpin RNAs that areprocessed into UiRNAs by cellular enzymes, (5) viral vectors that directthe transcription of double-stranded UiRNAs, and (6) viral vectors thatdirect the transcription of small single-stranded hairpin RNAs that areprocessed into UiRNAs by cellular enzymes, etc.

The compositions are prepared as a pharmaceutical formulation suitablefor administration into a patient or infected organism. Accordingly, thepresent invention also extends to pharmaceutical compositions,medicaments, drugs or other compositions containing one or more of thetherapeutic agent in accordance with the present invention.

In the pharmaceutical composition, an active compound identified inaccordance with the present invention can be in any pharmaceuticallyacceptable salt form. As used herein, the term “pharmaceuticallyacceptable salts” refers to the relatively non-toxic, organic orinorganic salts of the compounds of the present invention, includinginorganic or organic acid addition salts of the compound. Examples ofsuch salts include, but are not limited to, hydrochloride salts, sulfatesalts, bisulfate salts, borate salts, nitrate salts, acetate salts,phosphate salts, hydrobromide salts, laurylsulfonate salts,glucoheptonate salts, oxalate salts, oleate salts, laurate salts,stearate salts, palmitate salts, valerate salts, benzoate salts,naphthylate salts, mesylate salts, tosylate salts, citrate salts,lactate salts, maleate salts, succinate salts, tartrate salts, fumaratesalts, and the like. See, e.g., Berge, et al., J. Pharm. Sci., 66:1-19(1977).

For oral delivery, the active compounds can be incorporated into aformulation that includes pharmaceutically acceptable carriers such asbinders (e.g., gelatin, cellulose, gum tragacanth), excipients (e.g.,starch, lactose), lubricants (e.g., magnesium stearate, silicondioxide), disintegrating agents (e.g., alginate, Primogel, and cornstarch), and sweetening or flavoring agents (e.g., glucose, sucrose,saccharin, methyl salicylate, and peppermint). The formulation can beorally delivered in the form of enclosed gelatin capsules or compressedtablets. Capsules and tablets can be prepared in any conventionaltechniques. The capsules and tablets can also be coated with variouscoatings known in the art to modify the flavors, tastes, colors, andshapes of the capsules and tablets. In addition, liquid carriers such asfatty oil can also be included in capsules.

Suitable oral formulations can also be in the form of suspension, syrup,chewing gum, wafer, elixir, and the like. If desired, conventionalagents for modifying flavors, tastes, colors, and shapes of the specialforms can also be included. In addition, for convenient administrationby enteral feeding tube in patients unable to swallow, the activecompounds can be dissolved in an acceptable lipophilic vegetable oilvehicle such as olive oil, corn oil and safflower oil.

The active compounds can also be administered parenterally in the formof solution or suspension, or in lyophilized form capable of conversioninto a solution or suspension form before use. In such formulations,diluents or pharmaceutically acceptable carriers such as sterile waterand physiological saline buffer can be used. Other conventionalsolvents, pH buffers, stabilizers, anti-bacterial agents, ribonucleaseinhibitors, surfactants, and antioxidants can all be included. Forexample, useful components include sodium chloride, acetate, citrate orphosphate buffers, glycerin, dextrose, fixed oils, methyl parabens,polyethylene glycol, propylene glycol, sodium bisulfate, benzyl alcohol,ascorbic acid, and the like. The parenteral formulations can be storedin any conventional containers such as vials and ampoules.

Routes of topical administration include nasal, bucal, mucosal, rectal,or vaginal applications. For topical administration, the activecompounds can be formulated into lotions, creams, ointments, gels,powders, pastes, sprays, suspensions, drops and aerosols. Thus, one ormore thickening agents, humectants, and stabilizing agents can beincluded in the formulations. Examples of such agents include, but arenot limited to, polyethylene glycol, sorbitol, xanthan gum, petrolatum,beeswax, or mineral oil, lanolin, squalene, and the like. A special formof topical administration is delivery by a transdermal patch. Methodsfor preparing transdermal patches are disclosed, e.g., in Brown, et al.,Annual Review of Medicine, 39:221-229 (1988), which is incorporatedherein by reference.

Subcutaneous implantation for sustained release of the active compoundsmay also be a suitable route of administration. This entails surgicalprocedures for implanting an active compound in any suitable formulationinto a subcutaneous space, e.g., beneath the anterior abdominal wall.See, e.g., Wilson et al., J. Clin. Psych. 45:242-247 (1984). Hydrogelscan be used as a carrier for the sustained release of the activecompounds. Hydrogels are generally known in the art. They are typicallymade by crosslinking high molecular weight biocompatible polymers into anetwork that swells in water to form a gel like material. Preferably,hydrogels is biodegradable or biosorbable. For purposes of thisinvention, hydrogels made of polyethylene glycols, collagen, orpoly(glycolic-co-L-lactic acid) may be useful. See, e.g., Phillips etal., J. Pharmaceut. Sci. 73:1718-1720 (1984).

The active compounds can also be conjugated, to a water-solublenon-immunogenic non-peptidic high molecular weight polymer to form apolymer conjugate. For example, an active compound is covalently linkedto polyethylene glycol to form a conjugate. Typically, such a conjugateexhibits improved solubility, stability, and reduced toxicity andimmunogenicity. Thus, when administered to a patient, the activecompound in the conjugate can have a longer half-life in the body, andexhibit better efficacy. See generally, Burnham, Am. J. Hosp. Pharm.,15:210-218 (1994). PEGylated proteins are currently being used inprotein replacement therapies and for other therapeutic uses. Forexample, PEGylated interferon (PEG-INTRON A®) is clinically used fortreating Hepatitis B. PEGylated adenosine deaminase (ADAGEN®) is beingused to treat severe combined immunodeficiency disease (SCIDS).PEGylated L-asparaginase (ONCAPSPAR®) is being used to treat acutelymphoblastic leukemia (ALL). It is preferred that the covalent linkagebetween the polymer and the active compound and/or the polymer itself ishydrolytically degradable under physiological conditions. Suchconjugates known as “prodrugs” can readily release the active compoundinside the body. Controlled release of an active compound can also beachieved by incorporating the active ingredient into microcapsules,nanocapsules, or hydrogels generally known in the art.

Liposomes can also be used as carriers for the active compounds of thepresent invention. Liposomes are micelles made of various lipids such ascholesterol, phospholipids, fatty acids, and derivatives thereof.Various modified lipids can also be used. Liposomes can reduce thetoxicity of the active compounds, and increase their stability. Methodsfor preparing liposomal suspensions containing active ingredientstherein are generally known in the art. See, e.g., U.S. Pat. No.4,522,811; Prescott, Ed., Methods in Cell Biology, Volume XIV, AcademicPress, New York, N.Y. (1976).

The active compounds can also be administered in combination withanother active agent that synergistically treats or prevents the samesymptoms or is effective for another disease or symptom in the patienttreated so long as the other active agent does not interfere with oradversely affect the effects of the active compounds of this invention.Such other active agents include but are not limited toanti-inflammation agents, antiviral agents, antibiotics, antifungalagents, antithrombotic agents, cardiovascular drugs, cholesterollowering agents, anti-cancer drugs, hypertension drugs, and the like.

Generally, the toxicity profile and therapeutic efficacy of thetherapeutic agents can be determined by standard pharmaceuticalprocedures in cell models or animal models, e.g., those provided inSection 9, 10, 11 and 13, above. As is known in the art, the LD50represents the dose lethal to about 50% of a tested population. The ED50is a parameter indicating the dose therapeutically effective in about50% of a tested population. Both LD50 and ED50 can be determined in cellmodels and animal models. In addition, the IC50 may also be obtained incell models and animal models, which stands for the circulating plasmaconcentration that is effective in achieving about 50% of the maximalinhibition of the symptoms of a disease or disorder. Such data may beused in designing a dosage range for clinical trials in humans.Typically, as will be apparent to skilled artisans, the dosage range forhuman use should be designed such that the range centers on the ED50and/or IC50, but significantly below the LD50 obtained from cell oranimal models.

It will be apparent to skilled artisans that therapeutically effectiveamount for each active compound to be included in a pharmaceuticalcomposition of the present invention can vary with factors including butnot limited to the activity of the compound used, stability of theactive compound in the patient's body, the severity of the conditions tobe alleviated, the total weight of the patient treated, the route ofadministration, the ease of absorption, distribution, and excretion ofthe active compound by the body, the age and sensitivity of the patientto be treated, and the like. The amount of administration can also beadjusted as the various factors change over time.

EXAMPLES

The present invention is described in further detail by way of thefollowing illustrative examples. However, it is not intended that thepresent invention be limited to the examples provided.

1. Determining What Regions of the West Nile Virus Genome Represent theBest Target Sequences for an RNAi-Based Approach to Blocking ViralInvasion, Replication, Packaging or Egress in the Mosquito Vector, Aedesaegypti.

West Nile virus (WNV) belongs to the family Flaviviridae; a family thatcomprises more than 60 viruses, many of which are important humanpathogens. WNV primarily infects birds, but occasionally also infectshumans and horses. Like denge virus, a closely related fellowflavivirus, WNV is transmitted to humans by the bite of the Aedesaegypti mosquito. Advantageously, A. aegypti cells (ATCC CCL-125) can begrown in culture, and are susceptible to WNV infection in vitro. WNVreplicates in the cytoplasm of infected cells and has a positive strandRNA genome of about 11 kb that encodes a single polyprotein. Thesefeatures make WNV a useful model system with which to study all membersof the family Flaviviridae.

Recently, Caplen and colleagues at the National Institutes of Healthproposed that inhibition of viral gene expression within an insect hostcould be used to block virus replication and subsequent transmission ofthe pathogen to humans. Using Ades albopictus C6/36 cells, and SemlikiForest virus replicons, Caplen and coworkers demonstrated thatdsRNA-triggered inhibition of gene expression and viral replicationcould indeed be accomplished in mosquito cells (Caplen et al., Mol.Ther. 6:243-(2002)).

The methods of the present invention can be applied in order todetermine which regions of the West Nile virus genome represent the bestsequences for an RNAi-based approach to blocking viral invasion,replication, packaging or egress in the mosquito vector, Aedes aegyptiin an array-based approach as follows.

Prepare a set of expression vectors, each containing an expressioncassette that directs the expression of a chimeric RNA transcriptencoding a short polypeptide representing a specific portion of about 10to 20 amino acid residues of the West Nile virus polyprotein, and aUtRNA. Preferably the set of vectors direct the expression of a set offusion proteins comprising polypeptides that match the sequence of theviral polyprotein at regularly spaced intervals, and a carboxyl-terminalc-myc epitope tag. The set of expression vectors are designed to directthe transcription of the encoded chimeric RNA transcripts in insectcells, using appropriate promoter sequences, etc.

Using this set of expression vectors, and the techniques of Sabatinidisclosed in U.S. Pat. No. 6,544,790, prepare a matched pair ofmicroarrays of A. aegypti cells (ATCC CCL-125) wherein specific clustersof cells are transfected with the various expression vector in the set.After 12-48 hours, introduce a UiRNA into the cells of one transfectedcell microarray, and leave the second transfected cell microarray as anuntreated control. The UiRNA will induce a primary RNAi response bytargeting the UtRNA of the chimeric RNA transcripts, but secondarysiRNAs corresponding to sequences in the viral genome will be generatedby a secondary RNAi response. After an additional 12-48 hours, exposethe cells of both transfected cell microarrays to West Nile virussuspended in the culture medium. After 12-48 hours, observe the clustersof cells in the two microarrays, and determine which cell clusters ineither microarray show no signs of infection by the virus. Cell clustersthat show no sign of infection in the UiRNA-treated microarray, but areinfected in the untreated microarray indicate cells in which RNAiinduced by specific secondary siRNAs effectively blocked viral invasion,replication, packaging or egress. The address of the cells within themicroarray reveals what expression vector was used for reversetransfection of those cells, and the viral sequence contained within theexpression cassette within the expression vector corresponds to apreferred siRNA target sequence. If the viral gene identity in theidentified cells in not readily apparent, isolate the cells and analyzethe DNA in the expression cassette to determine the identity of thegene.

2. Determining or Confirming What Drug Metabolizing Enzymes (DME) areResponsible for the Detoxification of a Toxicant.

Prepare a set of expression vectors, each containing an expressioncassette that directs the expression of a chimeric RNA transcriptencoding a particular DME, and a UtRNA. Preferably the set of vectorscarry an inducible or derepressible promoter such that the transcriptionof the encoded chimeric RNA transcripts can be manipulated in humancells.

Using this set of expression vectors, and the techniques of Sabatinidisclosed in U.S. Pat. No. 6,544,790, prepare a matched pair ofmicroarrays of human cells wherein specific clusters of cells aretransfected with the various expression vector in the set. After 12-48hours, introduce a UiRNA into the cells of one transfected cellmicroarray, and leave the second transfected cell microarray as anuntreated control. The UiRNA will induce an RNAi response by targetingthe UtRNA in all of the chimeric RNA transcripts. After an additional12-48 hours, expose the cells of both transfected cell microarrays to atoxicant suspended in the culture medium. After 12-48 hours, observe theclusters of cells in the two microarrays, and determine which cellclusters in either microarray are resistant to the toxicant. Cellclusters that are resistant to the toxicant in the untreated controlarray, but show signs of intoxication in the UiRNA-treated microarray,are cells containing expression vectors encoding a DME that can catalyzethe detoxification of the toxicant under study.

All publications, patents and patent application publications citedabove are herein incorporated by reference to the same extent as if eachindividual publication, patent or patent application publication wasspecifically and individually indicated to be incorporated by reference.The mere mentioning of the publications and patent applications does notnecessarily constitute an admission that they constitute prior art tothe instant invention or application.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

1. A method of reducing the endogenous expression of a plurality of geneproducts in cells or organisms that exhibit transitive RNA interferencecomprising: providing a plurality of expression cassettes that directthe expression of recombinant transcripts, each transcript having aunique nucleotide sequence encoding an individual gene product and auniversal RNA interference target sequence that is 3′ of, and adjacentto, said unique nucleotide sequence encoding an individual gene product,creating a plurality of transfected test cells by introducing saidexpression cassettes into cells capable of transcribing and translatingthe encoded individual gene products, and introducing an RNA into saidtransfected test cells, said RNA being capable of inducing RNAinterference by specifically targeting the universal RNA interferencetarget sequence shared by the recombinant transcripts; whereupon theexpression of all recombinantly expressed gene products is reduced by aprimary RNA interference response, and the expression of homologousendogenously expressed gene products is reduced by a transitive RNAinterference response.
 2. The method of claim 1, wherein the step ofintroducing said RNA is by way of introducing a DNA that directs the invivo transcription of said RNA.
 3. The method of claim 2, wherein theRNA transcribed in vivo is a small hairpin RNA.
 4. The method of claim1, wherein the step of introducing said RNA is by way of introducing anRNA synthesized in vitro.
 5. The method of claim 4, wherein the RNAsynthesized in vitro is a small single-stranded hairpin RNA.
 6. Themethod of claim 4, wherein the RNA synthesized in vitro is adouble-stranded siRNA.
 7. The method of claim 1, wherein said universalRNA interference target sequence is located in the 3′ untranslatedregion of the recombinant transcript.
 8. The method of claim 1, whereinsaid universal RNA interference target sequence encodes a peptide, iscloned in frame with the 3′ end of said nucleotide sequence encoding anindividual gene product, and results in the expression of a fusionprotein.
 9. A method of treating a disease in plants caused by aninfectious agent comprising the steps of: administering to a plant inneed of such treatment an expression cassette comprising a nucleotidesequence encoding a gene product of said infectious agent and auniversal RNA interference target sequence, and introducing into theplant an RNA that induces RNA interference of expression of said geneproduct of said infectious agent by targeting said universal RNAinterference target sequence.
 10. The method of claim 9, wherein thestep of introducing said RNA that induces RNA interference is by way ofintroducing a DNA that directs the in vivo transcription of the RNA. 11.The method of claim 10, wherein the RNA transcribed in vivo is a smallhairpin RNA.
 12. The method of claim 9, wherein the step of introducingsaid RNA that induces RNA interference is by way of introducing an RNAsynthesized in vitro.
 13. The method of claim 12, wherein the RNAsynthesized in vitro is a small single-stranded hairpin RNA.
 14. Themethod of claim 12, wherein the RNA synthesized in vitro is adouble-stranded siRNA.
 15. The method of claim 12, wherein the RNAsynthesized in vitro is enzymatically synthesized.
 16. The method ofclaim 12, wherein the RNA synthesized in vitro is chemicallysynthesized.
 17. The method of claim 9, wherein said disease is a viralinfection and said infectious agent is the virus that causes said viralinfection.
 18. The method of claim 9, wherein said disease is a fungalinfection and said infectious agent is the fungus that causes saidfungal infection.
 19. The method of claim 9, wherein said disease iscaused by nematodes and said infectious agent is the nematode thatcauses the disease.
 20. A method of imparting on plants a resistance toa pathogen comprising the steps of: administering to a plant in need ofresistance to a pathogen an expression cassette that directs theexpression of a chimeric RNA comprising a nucleotide sequence encoding agene product of said pathogen, or a portion thereof, operably linked toa universal RNA interference target sequence, and introducing into theplant an RNA that induces RNA interference of expression of said geneproduct of said pathogen by targeting said universal RNA interferencetarget sequence.